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10 appendices

APPENDIX A: Data/Specimen Collection, Storage and Disposition Procedures Intended use of samples - translational studies on tumor samples:
Tubulin immunostaining: Immunohistochemistry (IHC) will be performed on formalin fixed, paraffin embedded sections using microwave antigen retrieval. An automated immunohistochemical staining procedure will be used according to manufacturer's recommendations (TechMate 1000™, Ventana, Tucson, AZ). Primary antibodies will be DO-7 for p53 (Dako, Santa Barbara, CA), and DM1A, a monoclonal anti-a-tubulin (Sigma, St Louis, MO). PARP activation: Fresh patient core biopsies will be Dounce homogenized at 22ºC in hypotonic lysis buffer [1 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-40, 20 mM Tris-HCl, pH 6.8, containing protease inhibitors] and vortexed vigorously. The samples will be pulse sonicated to clarity prior to centrifugation for 1 min at 1000 rpm to remove unlysed debris. Total protein concentrations in the lysates will be determined using the Biorad assay and equal amounts of protein will be separated on 4-15% gradient SDS-PAGE gels for each pair of pre- and post-treatment samples. To assess apoptosis, PARP cleavage will be assayed by probing the Western blot with a rabbit polyclonal anti-PARP
antibody that detects both the full length and the 85 kDa cleavage product (SC-7150 [H250] Santa Cruz, CA). Anti Fractin antibody may also be used. Immunoblots probed with either of these antibodies will also be simultaneously or sequentially probed with an antibody to either actin or GAPDH, as loading controls for each pair. cDNA Micro-array: Biopsy material that will be used for micro-array analyses will be flash-frozen as soon as practically possible after obtaining the sample (< 20 minutes). The biopsy will be flash-frozen using either liquid nitrogen or a dry ice ethanol bath and then stored at –70 °C to –80 °C. Total RNA will be prepared from frozen biopsy tissue using RNeasy® Kits (Qiagen, Valencia, CA) following the manufactures instructions. Micro-arrays will be used to measure the steady state expression level of nearly all known human genes in the fresh-frozen biopsies. Approximately 3 - 10 mg of total RNA will be used to generate fluorescent-labeled cDNA for hybridization to the Human Genome U95 gene set (Affymetrix, Inc. Santa Clara CA). The complete Human U95 gene set contains 60,000 expression elements that represent approximately 12,000 full length genes and 50,000 ESTs.  
Collection, handling, and storing of blood specimens for tubulin polymerization assay using PBMCs: (1) Collection: Blood samples are to be collected from an indwelling catheter or by direct venipuncture. Each blood sample should be collected into a pre-labeled 8 mL BectonDickinson CPT Vacutainer® tube. (2) Handling: Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant. Tubes should remain at room temperature and be centrifuged for 30 minutes at approximately 1700 x g at 20°C within 15 minutes of collection. The PBMCs should be transferred to a separate polypropylene tube and washed twice with 10 mL of ice-cold phosphate buffered saline (PBS). Following each washing, cells should be separated by centrifugation at 1700 x g for 10 minutes. Following the second washing, remove as much liquid as possible. The tube may be inverted on a paper towel for a few minutes to remove residual liquid, provided the cell pellet does not become dislodged from the tube bottom. (3) Storing: The PBMC cellular pellet should be stored at -70 to -80°C until assayed. Samples should be labelled. Each label will have the study number, sample matrix, and nominal sample time. Once affixed on tubes, the labels should be wrapped completely by applying protective transparent tape for protection during handling, and storage. The actual time, as well as the date, for each sample should be recorded.
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tracking and storage of samples

When initially collected, all specimens should be labeled with the patient's name, hospital number, date and time of collection. Once the samples reach the laboratory they will be appropriately labeled and coded without names. The information on the subjects' names and ID numbers will be kept in a log book in a locked cabinet.  
At this time (11/2008) the samples are recorded in a laboratory notebook in room 12N220. The information includes the patient's name, ID number, date of biopsy and whether the sample is pre- or post-ixabepilone administration. We intend to upgrade the tracking process to an electronic spreadsheet that contains the patient ID, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Samples remaining after the study completes will be stored in the same freezer in Dr. Fojo's lab and eventually transferred to Dr. Figg's core facility for possible future study as outlined below. The study consent contains a section asking patients for permission to keep the tissue for use in research to learn about, prevent, or treat cancer or other health problems.  
Material remaining after completion of correlative studies (with the exception of biopsy samples) will be transferred to the laboratory of Dr. William Figg to be entered into the Clinical Pharmacology Program's "Patient Sample Database Management System" (PSDMS) -
Labrador. This is a secure program that can only be accessed by authorized users in Dr. Figg's lab. PSDMS creates a unique barcode ID for every sample and sample box that cannot be traced back to patients without PSDMS access. These samples will be managed in accordance with the laboratory's policies and procedures. The data recorded for each sample includes the patient ID, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. There are patient demographics that can be obtained to correlate with the samples through the PSDMS system. For each samples, there are notes describing the processing method (delay in sample processing, storage conditions on the ward, etc.). Bar-coded samples are stored in bar-coded boxes in a locked freezer at either -20 or -85qC according to stability requirements. These freezers are located onsite in Dr. Figg's lab and offsite at NCI Frederick Central Repository Services (Fisher Bioservices) in Frederick, MD. Samples will be stored until requested by the researcher assigned to the protocol (however, those requests must come from a member of Dr. Figg's laboratory with PSDMS access/clearance). All requests are monitored and tracked in the PSDMS system. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol - that protocol is stored in the PSDMS system) and that any unused samples must be returned to Dr. Figg's laboratory.  
Guidelines for reporting loss or destruction to IRB: The PI will report any loss or destruction of samples to the IRB. Any new use of the samples, specimens, or data outside of this protocol will require prospective IRB review and approval. APPENDIX B: Study Calendar  
| EVALUATIONS REQUIRED PRE, DURING, AND POST THERAPY: ABCD                                                                                                                | E   | F   |    |    |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----|-----|----|----|
| Complete medical history                                                                                                                                                | X   |     |    |    |
| Interim medical history                                                                                                                                                 | X   | X   |    |    |
| Physical examination                                                                                                                                                    | X   | X   | X  |    |
| Weight and height                                                                                                                                                       | X   | X   |    |    |
| Vital signs                                                                                                                                                             | X   | X   | X  |    |
| ECOG performance status                                                                                                                                                 | X   | X   | X  | X  |
| Urine pregnancy test                                                                                                                                                    | X   |     |    |    |
| HIV Screen/Hepatitis B and C Screen                                                                                                                                     | X   |     |    |    |
| Staging laboratory evaluation                                                                                                                                           | X   | X   | X  |    |
| Routine laboratory evaluation                                                                                                                                           | X   | X   | X  |    |
| CBC with differential and platelet count                                                                                                                                | X   | X   | X  | X  |
| Staging imaging studies                                                                                                                                                 | X   | X   | X  |    |
| Biopsy & Research bloods (if indicated)*                                                                                                                                | X   | X   |    |    |
| ____________________________________________________________________________ A: Within 4 weeks prior to initiating therapy B: Within 1 week prior to initiating therapy |     |     |    |    |  
Complete medical history X Interim medical history X X Physical examination X X X  
Weight and height X X  
Vital signs X X X ECOG performance status X X X X  
Urine pregnancy test X HIV Screen/Hepatitis B and C Screen X  
Staging laboratory evaluation X X X  
Routine laboratory evaluation X X X  
CBC with differential and platelet count X X X X  
Staging imaging studies X X X  
Biopsy & Research bloods (if indicated)* X X  
____________________________________________________________________________  
A: Within 4 weeks prior to initiating therapy  
B: Within 1 week prior to initiating therapy  
C: Weekly or twice weekly during period of anticipated neutropenia  
D: Every cycle  
E: Every 2 cycles (Imaging studies and chest radiograph every third cycle after cycle 6)  
F: Study termination. *Biopsies will be obtained after consent but before Day 1 dosing, and again on either Day 4 or Day 5. Biopsies are optional.  
Research Labs for tubulin polymerization will be obtained prior to the Cycle 1 Day 1 drug  
infusion, and 1, 8 and 24 hours after Day 1 infusion start, and on either Day 4 or 5, within 1 hour of the biopsy. The research labs will only be done in those patients who have biopsies performed.  
APPENDIX C: List of drugs that may have potential CYP3A4 interactions
(source: ctep.cancer.gov) [Updated on May 1, 2007]
When drugs classified as 'substrates' are co-administered with BMS-247550 there is the potential for higher concentrations of the 'substrate'. When BMS-247550 is coadministered with compounds classified as 'inhibitors', increased plasma concentrations of BMS-247550 is the potential outcome. The co-administration of 'inducers' would potentially lower plasma BMS-247550 concentrations. Only major substrates and effective inducers are listed.
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list of drugs that may

| CYP3A4 Substrates Albuterol Alfentanil Alprazolam Amiodarone Amlodipine Amprenavir Aprepitant Aripiprazole Atazanavir Atorvastatin Benzphetamine Bisoprolol Bortezomib Bosentan Bromazepam Bromocriptine Budesonide Buprenorphine Buspirone Busulfan Carbamazepine Cerivastatin Chlordiazepoxide Chloroquine Chlorpheniramine Cilostazol Cisapride Citalopram Clarithromycin Clobazam Clonazepam Clorazepate Cocaine Colchicine Conivaptan Cyclophosphamide Cyclosporine Dantrolene Dapsone Dasatinib (1) Delavirdine Diazepam Dihydroergotamine  Diltiazem  Disopyramide  Docetaxel Doxepin Doxorubicin Doxycycline Efavirenz Eletriptan Enalapril Eplerenone Ergoloid mesylates Ergonovine Ergotamine Erythromycin Escitalopram Estradiol Estrogens, conj., synthetic Estrogens, conj., equine Estrogens, conj., esterified Estrone Estropipate Ethinyl estradiol Ethosuximide Etoposide Exemestane Felbamate Felodipine Fentanyl Flurazepam Flutamide Fluticasone Fosamprenavir Gefitinib Haloperidol Ifosfamide Imatinib Indinavir Irinotecan Isosorbide Isosorbide dinitrate Isosorbide mononitrate   | Isradipine  Itraconazole Ketamine  Ketoconazole Lansoprazole Letrozole Levonorgestrel Lidocaine Losartan Lovastatin Medroxyprogesterone  Mefloquine Mestranol Methadone Methylergonovine Methysergide Miconazole Midazolam Miglustat Mirtazapine Modafinil Montelukast Moricizine Nateglinide Nefazodone Nelfinavir Nevirapine Nicardipine Nifedipine Nimodipine Nisoldipine Norethindrone Norgestrel Ondansetron Paclitaxel Pergolide Phencyclidine Pimozide Pipotiazine Primaquine Progesterone Quetiapine   | Quinidine Rabeprazole  Ranolazine Repaglinide Rifabutin Ritonavir Salmeterol Saquinavir Sibutramine Sildenafil Simvastatin Sirolimus Spiramycin Sufentanil Sunitinib Tacrolimus Tamoxifen Tamsulosin Telithromycin Teniposide Tetracycline Theophylline Tiagabine Ticlopidine Tipranavir Tolterodine Toremifene Trazodone Triazolam Trimethoprim Trimipramine Troleandomycin Vardenafil Venlafaxine Verapamil Vinblastine Vincristine Vinorelbine Zolpidem Zonisamide Zopiclone   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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list of drugs that may

| CYP3A4 Inhibitors Acetominophen Acetazolamide Amiodarone Amlodipine Amprenavir Anastrozole Aprepitant Atazanavir Atorvastatin Azelastine Azithromycin Betamethasone Bortezomib Bromocriptine Caffeine Cerivastatin Chloramphenicol Chlorzoxazone Cimetidine Ciprofloxacin Cisapride Clarithromycin Clemastine Clofazimine Clotrimazole Clozapine Cocaine Conivaptan Cyclophosphamide Cyclosporine Danazol Dasatinib (1) Delavirdine Desipramine Dexmedetomidine Diazepam   | Diclofenac Dihydroergotamine  Diltiazem Disulfiram Docetaxel Doxorubicin Doxycycline Drospirenone Efavirenz Enoxacin Entacapone Ergotamine Erythromycin Ethinyl estradiol Etoposide Felodipine Fentanyl Fluconazole Fluoxetine Fluvastatin Fluvoxamine Fosamprenavir Glyburide Grapefruit juice (2) Haloperidol Hydralazine Ifosfamide Imatinib Indinavir Irbesartan Isoniazid Isradipine Itraconazole Ketoconazole Lansoprazole Lidocaine   | Lomustine Losartan  Lovastatin Mefloquine Mestranol Methadone Methimazole Methoxsalen Methylprednisolone Metronidazole Miconazole Midazolam Mifepristone Mirtazapine Mitoxantrone Modafinil Nefazodone Nelfinavir Nevirapine Nicardipine Nifedipine Nisoldipine Nizatidine Norfloxacin Olanzapine Omeprazole Orphenadrine Oxybutynin Paroxetine Pentamidine Pergolide Phencyclidine Pilocarpine Pimozide Pravastatin Prednisolone   | Primaquine Progesterone Propofol  Propoxyphene Quinidine Quinine Quinupristin Rabeprazole Ranolazine Risperidone Ritonavir Saquinavir Selegiline Sertraline Sildenafil Sirolimus Sulconazole Tacrolimus Tamoxifen Telithromycin Teniposide Testosterone Tetracycline Ticlopidine Tranylcypromine Trazodone Troleandomycin Valproic acid Venlafaxine Verapamil Vinblastine Vincristine Vinorelbine Voriconazole Zafirlukast Ziprasidone   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| CYP3A4 Inducers Aminoglutethimide Carbamazepine Fosphenytoin Nafcillin                                                                                                                                                                                                                                                                                                                                                                                                     | Nevirapine Oxcarbazepine Pentobarbital Phenobarbital                                                                                                                                                                                                                                                                                                                                                                                         | Phenytoin Primidone Rifabutin Rifampin                                                                                                                                                                                                                                                                                                                                                                                              | Rifapentine St. John's wort (3)                                                                                                                                                                                                                                                                                                                                                                                                          |
| When drugs classified as 'substrates' are co-administered with (Study Agent), there is the potential for higher concentrations of the 'substrate'.                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                          |  
When drugs classified as 'substrates' are co-administered with *(Study Agent)*, there is the potential for higher concentrations of the 'substrate'.  
When *(Study Agent)* is co-administered with compounds classified as 'inhibitors', increased plasma concentrations of *(Study Agent)* is the potential outcome. The co-administration of 'inducers' would potentially lower plasma *(Study Agent)* concentrations. Note: Adapted from Cytochrome P450 Enzymes: Substrates, Inhibitors, and Inducers. In: Lacy CF, Armstrong LL, Goldman MP,
Lance LL eds. Drug Information Handbook 15TH ed. Hudson, OH; LexiComp Inc. 2007: 1899-1912.  
Only major substrates and effective inducers are listed. Additional information for drug interactions with cytochrome P450 isoenzymes can be found at http://medicine.iupui.edu/flockhart/. (1) Investigator's Brochure: Dasatinib (BMS 354825). Bristol-Myers Squibb. October 2006.  
(2) Malhotra *et al*. (2001). Clin Pharmacol Ther. 69:14-23.  
(3) Mathijssen *et al*. (2002). J Natl Cancer Inst. 94:1247-1249.  
Frye *et al*. (2004). Clin Pharmacol Ther. 76:323-329.
APPENDIX D: Care of fingernails during treatment with Ixabepilone During the course of chemotherapy some patients have experienced pain in their fingertips, especially in their fingernails. This is especially true when they are trying to open a can or apply any pressure to their nails. Some patients have developed infections in their nail beds. Others have had their nails "lift off" over a period of time, as a new nail grows in.Sometimes the nails have streaks of color in them. This doesn't happen all at once, rather it happens gradually over the course of several months. We have developed some guidelines to help you care for your fingernails during chemotherapy. These techniques might help prevent or minimize the problems.
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prevention

1. Minimize hand and nail trauma. 2. Avoid repeated hand washing or soaking, for example, wear waterproof gloves when washing dishes.  
3. Avoid nail polish and solvents. 4. Avoid using instruments to dig under nails.  
5. Avoid exposing nails to direct sunlight (this has been a problem with a different chemotherapy. In that case, avoiding the sunlight helped).  
6. Wear gloves in the winter and sunscreen (containing titanium or iron oxide) on nails in the summer.  
7. Dry hand/nails thoroughly whenever they become wet. Some patients find a blow dryer is very helpful to dry the nails.  
8. Wear gloves when gardening or in situations that your nails might be exposed to dirt or solvents.  
Treatment 1. Signs of infections include nail discoloration, pus forming under the nail, or pus draining from the nail. If possible, the pus should be sent for "culture" to determine if any bacteria are present. This can be done by your local doctor or by the staff at the NIH.  
2. If you have an infection you can clean your nails with either hydrogen peroxide or dilute vinegar. You can put the solution in a small bowl and soak your fingertips for a few minutes, twice a day. Dry thoroughly when done.  
3. Some patients have found that trimming their nails very short has helped the infections clear more quickly.  
4. You may need antibiotics to treat the infection.