mithramycin-induced hepatotoxicity correlates
13.2.3 figure 3 effects of

With Abcb4 Snp  
7B  
  
13.2.7 Figure 7: Associations between ABCB4 and ABCB11 SNPs in mithramycin mediated hepatotoxicity in cancer patients  
  
  
  
| MTD? PK                                                                                                                                                                                                                                          | 50 ug/kg  tried " a surprising  factor was  severe  cumulative  toxicity -again  hemorrhage on  second course  with PLT >50 Not defined Not  defined   | -no evidence of  cumulative  hepatotox.                            |                                                      |                                                      |               |                                                                         |                                           |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------|------------------------------------------------------|------------------------------------------------------|---------------|-------------------------------------------------------------------------|-------------------------------------------|
| subsequent  cycles                                                                                                                                                                                                                               |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| Toxicity                                                                                                                                                                                                                                         | -one patient with bleeding time of 90  minutes                                                                                                         | -no deaths at 25 ug/kg with  continuous infusions                  | -Nausea, Vomiting, anorexia, restlessne irritability | -N/V - responded to anti-emetics NO  dose reductions | -uremia- 7/10 | -thrombocytopenia 1/10 (<150k) NO hemorrhage-withheld drug if  PLT<150k | -Isocitrate Dehydrogenase elevation  1/10 |
| 25 ug/kg Daily x 10  for cycle 1 Then A = Daily  x5 every  week for a  month Vs B = Q  week for 14  months Vs improved pain  and increased  activity (all regimens  effective) 10/10 with  Lebbin, D 75 1974 47-74 yrs 10 patients  with Paget's |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| -DEATH arterial bleed from  duodenal ulcer 1 Patient *thrombocytopenia 5%                                                                                                                                                                        |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| Hemorrhage- 3 deaths "usually related to  thrombocytopenia"                                                                                                                                                                                      | 1 patient removed for  "hepatotoxicity" not defined                                                                                                    | -Mean 2x AST/ALT                                                   |                                                      |                                                      |               |                                                                         |                                           |
| Moderate 18% Severe 5%                                                                                                                                                                                                                           |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| N/A 25 ug/kg Day 1 -Vomiting                                                                                                                                                                                                                     |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| Toxicity  In cycle 1                                                                                                                                                                                                                             | -Fever 9%                                                                                                                                              |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| -Phlebitis 11%                                                                                                                                                                                                                                   |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| 13.3 APPENDICES                                                                                                                                                                                                                                  | 13.3.1 Appendix 1: Mithramycin Clinical Experience Reported in the Literature                                                                          | M, W,F for  2 weeks  then one  dose  Qmonth 25 ug/kg  over 2-4 hrs |                                                      |                                                      |               |                                                                         |                                           |
| Daily as  tolerated  (up to 16) -a few got  24 ug/kg/day  as 24 hour  infusion                                                                                                                                                                   |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| Author Year Patient Age Disease Response rate Dose Schedule                                                                                                                                                                                      | 68                                                                                                                                                     |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| 25 (68 evaluated) 6 regressions 2 subjective  improvements 84 pts                                                                                                                                                                                | 13 pts 2SD 3PR 1CR (mithra +  interferon) CML                                                                                                          | Hypercalce mia 1992 27-74 (median 62)                              |                                                      |                                                      |               |                                                                         |                                           |
| Various 1 ESFT 1963 10-74 (10 yo  ESFT) 4 under 22                                                                                                                                                                                               |                                                                                                                                                        |                                                                    |                                                      |                                                      |               |                                                                         |                                           |
| Donaldson 86 1985 NONE Kofman 42 "Tx of  Disseminated."                                                                                                                                                                                          | Dutcher 72 1997 35-74 (median 51)                                                                                                                      | Thurlimann, B  79                                                  |                                                      |                                                      |               |                                                                         |                                           |  
| Discussi on of PK  data at  the end                                           |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
|-------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------|--------------------------------------------------|--------------------------------------------------|
| 3H drug  crosses  BBB ****                                                    |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
| MTD? PK subsequent  cycles Toxicity                                           | patient got a  second course  separated by a  month                                                                                                                                                                                                                                                                                                            | -stopped with  vomiting                                                               |                                                  |                                                  |
| Only one                                                                      | Not reported NB slow  infusion  attributed to  the low side  effects controlled with phenothiazine (5 pts)  OR droperidol -dizziness and headaches (poorly  responsive to antihistamines or  analgesia) -seizures in patient with cerebral mets - ONLY 2 required cessation of doses -NO LFT problems -thrombocytopenia in 1 -17 patients with nausea/vomiting |                                                                                       |                                                  |                                                  |
| -LFT elevations after 7 doses (3 Pt)  resolved                                | -hypocalcemia (4 pt) -Sepsis (1 pt) -severe bone pain (1 pt) -n/v 2 patients                                                                                                                                                                                                                                                                                   |                                                                                       |                                                  |                                                  |
| QOD for 3  weeks                                                              | Daily x 8 11 patients tolerated 8 day course -15 4-7 days -6 < 4days -none-10                                                                                                                                                                                                                                                                                  | -Anorexia/nausea 9 pts -Severe vomiting 5 pts -thrombocytopenia 4 pts -diarrhea 3 pts | -CNS symptoms 3 patients -thrombophlebitis 3 pts | -anemia (60%) mild to moderate -leukopenia (20%) |
| -otherwise limited toxicity                                                   |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
| 2 required held drug for  thrombocytopenia, N/V                               | -N/V in 58% of patients -mucositis                                                                                                                                                                                                                                                                                                                             |                                                                                       |                                                  |                                                  |
| Toxicity  In cycle 1                                                          | -FEW                                                                                                                                                                                                                                                                                                                                                           | Daily times  8 25 ug/kg  over 8  hours                                                | Daily times  21 ug/kg/day  over 6-8 hours        |                                                  |
| B1 = twice  weekly for  5 months                                              | doses over  8 to 14  days based  on symptoms                                                                                                                                                                                                                                                                                                                   |                                                                                       |                                                  |                                                  |
| 8 or 10                                                                       |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
| Author Year Patient Age Disease Response rate Dose Schedule                   | 69                                                                                                                                                                                                                                                                                                                                                             |                                                                                       |                                                  |                                                  |
| 25 ug/kg  + hydroxyur ea -1 returned to  chronic phase 9 Patients             | 25 ug/kg  over 12  hrs 12- no change 7 minor  regression 1 major  regression  (rectal CA) 32 patients fibrosarcoma Various -1                                                                                                                                                                                                                                  | 25 ug/kg  over 12  hours                                                              | 25                                               |                                                  |
| -tx with surgery  + radiation  +mithra                                        |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
| "quantitative  remission"                                                     | -6 stable disease                                                                                                                                                                                                                                                                                                                                              | -another rectal  cancer                                                               |                                                  |                                                  |
| 26 patients -4 pts with                                                       | 96 patients at  NIH 116 total 58 got mithra Anaplastic  gliomas Walker 67 1976 Median age  53 years                                                                                                                                                                                                                                                            |                                                                                       |                                                  |                                                  |
| 14                                                                            |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  |
| Abbreviated Title: Mithramycin for Cancer Stem Cells Version Date: 09/14/2018 | -1 alveolar  celled                                                                                                                                                                                                                                                                                                                                            | -1 leiomyosarc oma                                                                    |                                                  |                                                  |
| Various                                                                       | Ransohoff 71 1965 26-64 Glioblastom a (8 patients  with  improvemen ts)3 (3+), 2  (2+), 2 (2+)                                                                                                                                                                                                                                                                 |                                                                                       |                                                  |                                                  |
| 1991 28-57 CML in  blast crisis                                               | Baum, M 88 1968 42-83 (mean  61)                                                                                                                                                                                                                                                                                                                               | Sewell, I. A.69 1966 30-89 (mean 56)                                                  |                                                  |                                                  |
| Johnson, P.R.E 87                                                             |                                                                                                                                                                                                                                                                                                                                                                |                                                                                       |                                                  |                                                  | | -crosses  BBB but  should  not  (inhibits  MDR1)                                |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
|---------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|-----------------------------|----------------------|---------------------------------------------------------|
| of the  paper                                                                   |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| MTD? PK                                                                         | N/A N/A N/A                                                                                                                                    |                                                                                                                             |                             |                      |                                                         |
| ȝJGRVH                                                                                 |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| 50                                                                              |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| subsequent  cycles                                                              |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| Toxicity                                                                        | n/v. thrombocytopenia, hemorrhage,  hepatic damage (Transaminases, PT,  Alk Phos) Hemorrhage described in some detail Daily times  5 q 5-7 wks |                                                                                                                             |                             |                      |                                                         |
| -LFTs-mild 30%, moderate50% in  ALT/AST                                         | -hemorrhage into tumor bed in 3  patients- 2 DEATHS -chart summarizing tox                                                                     | -GI hemorrhage causing death in 1                                                                                           |                             |                      |                                                         |
| N/V, severe diarrhea in 3, LFTs                                                 | -thrombocytopenia in 1 patient                                                                                                                 |                                                                                                                             |                             |                      |                                                         |
| Not systematically reported  -thrombocytopenia seen                             |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| -thrombocytopenia                                                               | Thrombocytopenia Fever with infusion                                                                                                           |                                                                                                                             |                             |                      |                                                         |
| Toxicity  In cycle 1                                                            | -then 6 wks  later daily  times 12                                                                                                             | -then 6 wks  later daily  times 12                                                                                          | -83% got  11 doses          | -median is  21 doses | 50 x 1 then  60 ug/kg/day q  week x 8 NONE 50 ug/kg/dau |
| total of 6- 10 courses                                                          |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| -q3wks                                                                          |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| Author Year Patient Age Disease Response rate Dose Schedule                     | ug/kg/day  as 8-24  hour  infusion NOTE  ORAL  INACTIV E                                                                                       | 25, 30, 35,  40, 50,  60,70, 80,  90                                                                                        | (all at 40 ug/day  or more) |                      |                                                         |
| Embryonal CA  2 Chorio 1 Wilms 1 Breast 1                                       |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| 25                                                                              |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| -tx with surgery  + randomized to  mithra +/- radiation difference in  survival | ESFT 5 patients 1 durable CR 1973 Two  reported 21 and 23 yo                                                                                   | 5/26                                                                                                                        |                             |                      |                                                         |
| Abbreviated Title: Mithramycin for Cancer Stem Cells Version Date: 09/14/2018   | redundant  patients 3 ESFT  reported (2 repeats)                                                                                               | Variety of  adult  melanoma,  bronchogeni c CA,  hepatpma Spear 74 1963 58 patients (only 29  with  performance  status >50 |                             |                      |                                                         |
| A synovial                                                                      |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| Variety                                                                         |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |
| -no                                                                             | ? of  Kofman, s 70 1964 22 10 15 Much  younger  3 16 yo 2 23 yo Curreri, AR 68 1960 36 pts no  age                                             |                                                                                                                             |                             |                      |                                                         |
| Kofman  "metastatic  ewing;s 73                                                 |                                                                                                                                                |                                                                                                                             |                             |                      |                                                         |  
70  
| MTD? PK subsequent  cycles Toxicity  Q month Contraindicated: thrombocytopenia,  coagulopathy, poor bone marrow,  follow PLTs, PT and bleeding time -hemorrhagic syndrome is dose  related if 30 ug/kg/day or less 10 or  fewer bleeds –drug mortality rate is  1.6% IF >30 12% bleeding with mortality  rate of 5.7% COMMON: Anorexia, N/V, stomatitis LESS COMMON: Fever, drowsiness, lethargy, malaise,  H/A, depression, phlebitis, flushing,  skin arash LABS: Thrombocytopenia, leucopenia (6%),  increased clotting, bleeding time,  abnormal clot retraction, LFTs, AP,  LDH, bili Renal, Increased BUN, CR and  proteinuria Hypocalcemia, hypophosphatemia,  hypokalemiaToxicity  In cycle 1 Author Year Patient Age Disease Response rate Dose Schedule ug/kg/day  over 12  hours  daily x 8 q month dilute in  1L of d5  and give  over 4- 6hrs 25 Testicular  CA 1971 1600  patients Package insert  Pfizer 66   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| 13.3.2 Appendix 2: Study Calendar                                                                      | Cycles 1 and 2   |        |       |       |       |       |       |    |     |
|--------------------------------------------------------------------------------------------------------|------------------|--------|-------|-------|-------|-------|-------|----|-----|
| Research Eligibility  Evaluation/Baseline                                                              | Week 1           | Week 2 |       |       |       |       |       |    |     |
|                                                                                                        | day 1            | day 2  | day 3 | day 4 | day 5 | day 6 | day 7 |    |     |
| Mithramycin                                                                                            | x                | x      | x     | x     | x     | x     | x     |    |     |
| Histologic path  confirmation                                                                          | x                |        |       |       |       |       |       |    |     |
| ABCB4 and ABCB11 status                                                                                | X                |        |       |       |       |       |       |    |     |
| Informed consent                                                                                       | x                |        |       |       |       |       |       |    |     |
| Medical history                                                                                        | x                |        |       |       |       |       |       |    |     |
| Concurrent meds                                                                                        | x                |        |       |       |       |       |       |    |     |
| Physical examination                                                                                   | x                | xi     |       |       |       |       |       |    |     |
| Performance status                                                                                     | x                | xi     |       |       |       |       |       |    |     |
| General labs                                                                                           | x                | x      | x     | x     | x     | x     | x     | x  | xxb |
| Serum bile acids (total and  fractionated)                                                             | x                | x      | x     |       |       |       |       |    |     |
| Coagulation labs                                                                                       | x                | x      | xc    |       |       |       |       |    |     |
| Pregnancy test                                                                                         | x                |        |       |       |       |       |       |    |     |
| Cardiac function tests                                                                                 | x                |        |       |       |       |       |       |    |     |
| Imaging studies incl liver  Elastograpy by ultrasound                                                  | x                |        |       |       |       |       |       |    |     |
| Adverse events  Tumor biopsy                                                                           | x                |        |       |       |       |       |       |    |     |
| Research labs                                                                                          | xd               |        | xc    |       |       |       |       |    |     |
| PK                                                                                                     | x                | xdf    | xdf   | xdf   |       | xdf   | xg    |    |     |
| EKG                                                                                                    | x                | xd     |       | xd    |       | xd    |       |    |     |
| Cycles 3 and beyond*                                                                                   |                  |        |       |       |       |       |       |    |     |
| Week 1                                                                                                 | Week 2           |        |       |       |       |       |       |    |     |
| day 1                                                                                                  | day 2            | day 3  | day 4 | day 5 | day 6 | day 7 |       |    |     |
| Mithramycin                                                                                            | x                | x      | x     | x     | x     | x     | x     |    |     |
| Histologic path confirmation  Informed consent  Medical history  Concurrent meds  Physical examination | xi               |        | 72    |       |       |       |       |    |     |  
| Abbreviated Title: Mithramycin for Cancer Stem Cells Version Date: 09/14/2018                                                                                                                                                                                                                                                                                                                                                                                                    | Cycles 3 and beyond*   |       |       |       |       |       |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|-------|-------|-------|-------|-------|
| Week 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Week 2                 |       |       |       |       |       |
| day 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | day 2                  | day 3 | day 4 | day 5 | day 6 | day 7 |
| Performance status                                                                                                                                                                                                                                                                                                                                                                                                                                                               | xi                     |       |       |       |       |       |
| General labs                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | xh                     |       | x     | xa    |       |       |
| Coagulation labs                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | x                      | xc    |       |       |       |       |
| Serum bile acids (total and  fractionated)                                                                                                                                                                                                                                                                                                                                                                                                                                       | x                      | x     | xe    |       |       |       |
| (course 1 only)                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                        |       |       |       |       |       |
| Pregnancy test  Cardiac function tests                                                                                                                                                                                                                                                                                                                                                                                                                                           | xe                     |       |       |       |       |       |
| Imaging studies including liver  elastography by ultrasound Adverse events  Tumor biopsy  Research labs                                                                                                                                                                                                                                                                                                                                                                          | xd                     |       | xc    |       |       |       |
| EKG                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | xd                     |       | xd    |       |       |       |
| a - once weekly b - twice weekly c - day 8 of cycle (same as day 1 of week 2) d -prior to dosing e - two weeks (plus 12 or minus 3 days ) after the end of each course (1 course = 3 cycles) f - end of infusion g - 24 and 48 hours after day 7 infusion h- CBC with differential only i - within 4 days prior to the start of each cycle *Patients will continue to receive treatment until criteria for removal from protocol or off-study criteria are met (See section 3.6) |                        |       |       |       |       |       |  
| initials Target Time Actual Time EKG  Time                                          | 3 h post-start of  infusion n/a Just prior to the end  of the 6 hour infusion n/a 0.25 h post-infusion n/a 0.5 h post-infusion n/a 1 h post-infusion n/a 2 h post-infusion n/a 3 h post-infusion n/a 5 h post-infusion n/a 7 h post-infusion n/a 9-12 h post-infusion n/a   | PK 12 pre dose n/a PK 13 end of infusion n/a   | PK 14 pre dose n/a PK 15 end of infusion n/a   | PK 16 pre dose n/a PK 17 end of infusion n/a   | 24 hrs p/dose #7 n/a 24.5 hrs/p dose #7 n/a   | 48 hrs/p dose #7 n/a 48.5 hrs/p dose #7 n/a   |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
|-------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|------------------------------------------------|------------------------------------------------|-----------------------------------------------|-----------------------------------------------|---------|---------|---------|---------|---------|---------|---------|---------|---------|---------|--------|---------|---------|---------|
| 13.3.3 Appendix 3: SAMPLE Pharmacokinetic and EKG Worksheet for Mithramycin CYCLE 1 | TEL research                                                                                                                                                                                                                                                                | sample # (2 10 mL red top  tubes & 4 10 mL     | purple top tubes)                              |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| Hour (pre dose = within 1  hour prior to  infusion)                                 | 74                                                                                                                                                                                                                                                                          |                                                |                                                |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| PK 1 pre dose                                                                       |                                                                                                                                                                                                                                                                             |                                                |                                                |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| PK Sample # (two 3cc  lavender top  tube)                                           | PK 10 PK 11                                                                                                                                                                                                                                                                 | PK 18 PK 19                                    | PK 20 PK 21                                    |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| PK 2                                                                                | PK 3 PK 4 PK 5 PK 6 PK 7 PK 8 PK 9                                                                                                                                                                                                                                          |                                                |                                                |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| Abbreviated Title: Mithramycin for Cancer Stem Cells Version Date: 09/14/2018       | Coag reference  sample # (obtain kit from TEL)                                                                                                                                                                                                                              | (date)                                         | (date)                                         | (date)                                         | (date)                                        | (date)                                        | (date)  |         |         |         |         |         |         |         |         |         |        |         |         |         |
| C 1                                                                                 | n/a n/a                                                                                                                                                                                                                                                                     | n/a n/a                                        | n/a n/a                                        | n/a n/a                                        | n/a n/a                                       | n/a n/a                                       | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | R2 n/a | n/a n/a | n/a n/a | n/a n/a |
| C1D1                                                                                | C1D2                                                                                                                                                                                                                                                                        | C1D4                                           | C1D7                                           | C1D8                                           | C1D9                                          |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |
| R1                                                                                  |                                                                                                                                                                                                                                                                             |                                                |                                                |                                                |                                               |                                               |         |         |         |         |         |         |         |         |         |         |        |         |         |         |  
| initials Target Time Actual Time EKG  Time                                          | 3 h post-start of  infusion n/a Just prior to the end  of the 6 hour infusion n/a 0.25 h post-infusion n/a 0.5 h post-infusion n/a 1 h post-infusion n/a 2 h post-infusion n/a 3 h post-infusion n/a 5 h post-infusion n/a 7 h post-infusion n/a 9-12 h post-infusion n/a   | PK 12 pre dose n/a PK 13 end of infusion n/a   | PK 15 end of infusion n/a   | PK 17 end of infusion n/a   | 24 hrs p/dose #7 n/a 24.5 hrs/p dose #7 n/a   |         |         |         |         |         |         |         |         |         |         |        |         |
|-------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|-----------------------------|-----------------------------|-----------------------------------------------|---------|---------|---------|---------|---------|---------|---------|---------|---------|---------|--------|---------|
| 13.3.4 Appendix 4: SAMPLE Pharmacokinetic and EKG Worksheet for Mithramycin CYCLE 2 | TEL research                                                                                                                                                                                                                                                                | samples (2 10 mL red top  tubes & 4 10 mL      | purple top tubes)           |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |
| Hour (pre dose = within 1  hour prior to  infusion)                                 | 75                                                                                                                                                                                                                                                                          |                                                |                             |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |
| C 2 PK 1 pre dose                                                                   | PK 14 pre dose                                                                                                                                                                                                                                                              | PK 16 pre dose                                 |                             |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |
| PK Sample # (two 3cc  lavender top  tube)                                           | PK 10 PK 11                                                                                                                                                                                                                                                                 | PK 18 PK 19                                    |                             |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |
| PK 2                                                                                | PK 3 PK 4 PK 5 PK 6 PK 7 PK 8 PK 9                                                                                                                                                                                                                                          |                                                |                             |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |
| Coag reference # (obtain kit from  TEL)                                             | (date)                                                                                                                                                                                                                                                                      | (date)                                         | (date)                      | (date)                      | (date)                                        | (date)  |         |         |         |         |         |         |         |         |         |        |         |
| n/a n/a                                                                             | n/a n/a                                                                                                                                                                                                                                                                     | n/a n/a                                        | n/a n/a                     | n/a n/a                     | n/a n/a                                       | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | n/a n/a | R2 n/a | n/a n/a |
| C2D1                                                                                | C2D2                                                                                                                                                                                                                                                                        | C2D4                                           | C2D7                        | C2D8                        | C2D9                                          |         |         |         |         |         |         |         |         |         |         |        |         |
| n/a                                                                                 |                                                                                                                                                                                                                                                                             |                                                |                             |                             |                                               |         |         |         |         |         |         |         |         |         |         |        |         |  
  
  
n/a n/a  
  
  
n/a n/a
Instructions**PK Studies**Collect 6 mL blood in lavender top tubes. Blood has to be drawn from a site away from the site of infusion (cannot be drawn through the line used to infuse drug, or through another lumen of that line). Following blood collection, the tubes should be inverted several times to ensure mixing with the anticoagulant. Red top tubes should be kept at room temperature whereas lavender top tubes should be placed immediately on crushed ice. Samples should be transferred immediately to the TEL. Lavender top tubes should be centrifuged for 15 minutes atapproximately 1000 x g at 0-5 °C within 15 minutes of collection. The plasma should be transferred to separate pre-labeled screw-capped polypropylene tubes and stored at -80 °C until shipped to the analytical site. Total time from blood draw to freeze down of plasma should be less than 1 hour. **Research Labs**Samples will be collected in 2 red top and 4 lavender top tubes and transported on wet ice immediately to the TEL. Samples in lavender top tubes should be centrifuged at 1000 x g at 0-5oC for 15 minutes. Supernatent (plasma) should be transferred to separate pre-labeled screwcapped polypropylene tubes and stored at -80 °C for subsequent analysis. Pellet (PBMC) should be transferred to separate pre-labeled screwcapped polypropylene tubes and stored at -80 °C for subsequent analysis. Total time from blood draw to freeze down of plasma should be less than 1 hour. Red top tubes should be kept at room temperature for 1 hour. Thereafter, clot should be removed, and then samples centrifuged at 1000 x g at 0-5oC for 15 minutes. Supernatent (serum) should be transferred to separate pre-labeled screw-capped polypropylene tubes and stored at -80 °C for subsequent analysis. Total time from blood draw to freeze down of serum should be less than 2 hr. **Coagulation Studies**Samples will transported to the TEL lab on ice within 30 minutes. After centrifugation at 1000 x g at 0-5oC for 15 minutes, supernatant shouldbe filtered through a 0.22 micron filter, and transferred to separate pre-labeled screw-capped polypropylene tubes and stored at -80 °C for subsequent analysis.  
| initials Target Time Actual Time EKG  Time                                                    | 48 hrs/p dose #7 n/a 48.5 hrs/p dose #7 n/a   |
|-----------------------------------------------------------------------------------------------|-----------------------------------------------|
| Hour (pre dose = within 1  hour prior to  infusion) PK Sample # (two 3cc  lavender top  tube) | PK 20 PK 21                                   |
| Coag reference # (obtain kit from  TEL)                                                       |                                               |  
LabelingPrior to the infusion, the PK /research lab sheet and labels will be placed in the patient's room and a separate set of labels will be given to the TEL staff. The labels will list the patient identifiers and either "Cycle1" or "Cycle 2" as appropriate. As the RN (or designee) draws the bloods, she will place a label on the tube and write the sample reference \# on the label on the tube. When the TEL personnel or fellow processes the sample, she will label the storage tube with the label and will transcribe the corresponding sample reference \# on the label on the tube.**Tracking**Upon completion of the PK/Research sample draws, the research nurse will make 3 copies of the PK sheet. The original and one copy should be placed in the research binder. 2 copies should be given to the TEL personnel. The TEL will retain one copy for their records and will send the other copy with the PK samples to Dr. Figg's lab. Note: Prior to sending the PK sheets to Dr. Figg's lab, the patient name should be crossed out.  
13.3.5 Appendix 5: Sample Patient Log for Reactions/Symptoms NAME: ____________________________ Cycle: ___________________ Dates of last Mithramycin infusion: _____________  
| Reaction/Symptom                                                                                   | Start Date   | End Date   |
|----------------------------------------------------------------------------------------------------|--------------|------------|
| *Contact your study center team or local physician right away if you experience signs of bleeding. |              |            |  
*Contact your study center team or local physician right away if you experience signs of bleeding. This may include, but is not limited to, petechiae (red or purple spots on the skin), nosebleeds; bruising; or blood in urine, stool or vomit. Patient signature: __________________ Date:______________________ Reviewed by:_____________________ RN/MD Date:______________________  
13.3.6 Appendix 6: Sample Patient Log for Concomitant Medication NAME:____________________________ Cycle: ____________________ Dates of last Mithramycin infusion: _____________  
| Medication Taken                                                                                   | Start Date   | End Date   |
|----------------------------------------------------------------------------------------------------|--------------|------------|
| *Contact your study center team or local physician right away if you experience signs of bleeding. |              |            |  
*Contact your study center team or local physician right away if you experience signs of bleeding. This may include, but is not limited to, petechiae (red or purple spots on the skin), nosebleeds; bruising; or blood in urine, stool or vomit. Patient signature: __________________ Date:______________________ Reviewed by:_____________________ RN/MD Date:______________________
mithramycin-induced hepatotoxicity correlates
13.3.7 appendix 7 sample monitoring

Patient Name: _________________  
$$\mathbf{M}_{\mathrm{{\Phi}}}$$  
Dates and Doses of last Mithramycin: ___________________ Cycle: _________ Dear Dr. ____________________,
The patient named above is currently participating on a Phase I-II Mithramycin study at the National Cancer Institute at the National Institutes of Health in Bethesda, Maryland, and requires the following lab tests and evaluations between
____________________ and _____________________:
(date) (date)
o **History, physical exam**: Once Weekly
mithramycin-induced hepatotoxicity correlates
o weight once weekly

o **Labs**: (Circle one) Twice Weekly during Cycles 1-3 or Once Weekly after Cycle 3.  
CBC/Diff Chemistry Panel (include sodium, potassium, chloride, CO2, creatinine, glucose, BUN, albumin, ionized calcium**, magnesium, LDH, phosphorus, uric acid)
LFT *(include alk phos, ALT/AST, Total bilirubin, Direct bilirubin, Total protein)*
Coagulation profiles - *ONLY If clinically indicated:**
** only if covered by insurance Please FAX all lab results and medical summaries to Dr. David Schrump, M.D. or Tricia Kunst, R.N. at 301-451-6934 For any questions, please call 240-760-6239, 240-760-6234, or 240-760-6233 or email tricia.kunst@mail.nih.gov, or cara.kenney@nih.gov For urgent issues, contact the Attending Physician, Dr. David Schrump, through the NIH Operator at (301) 496-1211 ***,IWKHSDWLHQWH[SHULHQFHVDQ\VLJQVRIEOHHGLQJ*UDGHLQFOXGLQJHSLVWD[LVKHPDWXULDKHPDWHPHVLVRU** blood in the stool, the patient will require immediate comprehensive clinical evaluation and coagulation profile
(including PT/PTT/fibrinogen, thrombin time, PFA-100, D-dimer, Ca2+ and ionized calcium)
Please contact the NIH page operator at 301-496-1211 and ask for the Thoracic Surgery attending on call or Dr. David Schrump with any clinically significant bleed and support with appropriate blood product support.  
Bleeding that does not stop within 6 hours with appropriate treatment will be considered a dose limiting toxicity and further dosing of mithramycin will be discontinued.  
_______________________________________ _____________________________________
Physician Signature Print Name NIH/NCI/TGIB