MOLECULAR PHARMACOLOGY SUPPLEMENT

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1 MOLECULAR PHARMACOLOGY SUPPLEMENT Therapeutic targeting of a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter Sita Kugel Desmoulin, Lei Wang, Eric Hales, Lisa Polin, Kathryn White, Juiwanna Kushner, Mark Stout, Zhanjun Hou, Christina Cherian, Aleem Gangjee, and Larry H. Matherly Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, MI (SKD, LHM) Department of Oncology, Wayne State University School of Medicine, Detroit, MI (SKD, LP, EH, ZH, CC, LHM) Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI (LP, EH, ZH, CC, LHM) Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI (LHM) Department of Pediatrics, Children s Hospital of Michigan, Detroit, MI 48201(MS) Department of Internal Medicine, Division of Hematology-Oncology, Wayne State University School of Medicine, Detroit, MI (KW, JK). Division of Medicinal Chemistry, Graduate School of Pharmaceutical Science, Duquesne University, Pittsburgh, PA (LW, AG) 1

2 SUPPLEMENT Table 1S. Leukemia and Solid Tumor Cell Lines Used for Real-Time PCR. CELL LINE DISEASE ORIGIN SOURCE U937 AML Macrophage ATCC CTS AML M1 Peripheral blood Fuse, A. Kasumi-1 AML M2 Peripheral blood ATCC HL60 AML M2 Peripheral blood ATCC MV4-11 AML M5 Peripheral blood ATCC THP-1 AML M5 Peripheral blood ATCC AML-193 AML M5 Peripheral blood ATCC KG-1 AML M6 Bone marrow ATCC KG-1a AML M6 Bone marrow ATCC K562 1 AML M6 Bone marrow ATCC CMS AML M7 Peripheral blood Fuse, A. MEG-01 AML M7 Bone marrow ATCC CMK DS AML M7 Peripheral blood DSMZ CMY DS AML M7 Bone marrow Fuse, A. 697 BP-ALL Bone marrow DSMZ Nalm6 BP-ALL Peripheral blood DSMZ Uoc B4 BP-ALL CSF Findley, H. REH BP-ALL Unknown ATCC CCRF-CEM 1,2 T-cell ALL Peripheral blood ATCC MOLT4 1 T-cell ALL Peripheral blood DSMZ MOLT3 T-cell ALL Peripheral blood DSMZ HPB-ALL T-cell ALL Peripheral blood DSMZ TALL-1 T-cell ALL Bone marrow DSMZ DND 41 T-cell ALL Peripheral blood DSMZ ALL-SIL T-cell ALL Peripheral blood DSMZ Jurkat T-cell ALL Peripheral blood ATCC TE-85 Osteosarcoma Bone Peterson, W.D. HTB166 Ewing's sarcoma Bone ATCC MCF-7 1,2 Adenocarcinoma Breast ATCC MDA-MB231 1,2 Adenocarcinoma Breast ATCC MDA-MB435 1,2 Adenocarcinoma/Melanoma Breast/Skin? ATCC T-47D 1 Ductal carcinoma Breast ATCC KB Adenocarcinoma Cervix ATCC HeLa 2 Adenocarcinoma Cervix ATCC HCT-116 1,2 Colorectal adenocarcinoma Colon ATCC SW Colorectal adenocarcinoma Colon ATCC HCT15 1,2 Colorectal adenocarcinoma Colon ATCC Caco-2 Colorectal adenocarcinoma Colon ATCC BCPC-3 Adenocarcinoma Pancreas ATCC UCVA-1 Adenocarcinoma Pancreas Peterson, W.D. 786-O 1,2 Renal cell adenocarcinoma Kidney ATCC ACHN 1,2 Renal cell adenocarcinoma Kidney ATCC IGROV-1 1 Adenocarcinoma Ovary Ratnam, M. OVCAR-3 1,2 Adenocarcinoma Ovary ATCC SKOV-3 1 Adenocarcinoma Ovary ATCC HepG2 2 Hepatocellular carcinoma Liver ATCC 2

3 Hep3B 2 Hepatocellular carcinoma Liver ATCC Y79 Retinoblastoma Eye, retina ATCC SK-MEL5 1,2 Melanoma Skin ATCC SK-MEL-28 1 Melanoma Skin ATCC HTB139 Rabdomyosarcoma Muscle Peterson, W.D. SK-N-SH Neuroblastoma Brain ATCC SK-N-BE Neuroblastoma Brain ATCC SK-N-MC Neuroepithelioma Supraorbital area ATCC HT1080 Fibrosarcoma Connective tissue ATCC PC-3 1,2 Adenocarcinoma Prostate ATCC DU-145 1,2 Carcinoma Prostate ATCC H1650 Bronchoalveolar adenocarcinoma lung ATCC H2122 Adenocarcinoma/NSCLC lung ATCC H2030 Adenocarcinoma/NSCLC lung ATCC H1573 Adenocarcinoma lung ATCC H1781 Bronchoalveolar adenocarcinoma lung ATCC H3255 Adenocarcinoma/NSCLC lung Gazdar, A.F. A549 1 Carcinoma lung ATCC CRL5807 Bronchioalveolar carcinoma /NSCLC lung ATCC CRL5872 Adenocarcinoma/NSCLC lung ATCC CRL5810 1,2 Adenocarcinoma/NSCLC lung ATCC CRL5800 1,2 Adenocarcinoma/NSCLC lung ATCC H596 Adenosquamous carcinoma Lung ATCC NCI-H460 1,2 Large-cell carcinoma Lung ATCC H69 SCLC Lung ATCC H446 SCLC Lung ATCC H226 1 Pleural mesothelioma Pleura ATCC H2373 Pleural mesothelioma Pleura Pass, H.I. H2452 Pleural mesothelioma Pleura Pass, H.I. H2461 Pleural mesothelioma Pleura Pass, H.I. H2591 Pleural mesothelioma Pleura Pass, H.I. H2595 Pleural mesothelioma Pleura Pass, H.I. H2714/HP-1 Pleural mesothelioma Effusion Pass, H.I. 1 Cell lines of NCI60 cell line panel 2 Cell lines used in the report of Zhao, R. et al. (2004) 3

4 Table 2S. Real-time PCR Primers and Conditions. Table 2S. Real-time PCR Primers and Conditions. Gene Primer Sequences Probe Size (bp) Annealing ( C) PCFT RFC FRα GAPDH GenBank Accession Left: 5 -CTCATCCCGGCTGTTCTG-3 Right: 5 -CTGGAACTCGAGGTGAGGAT NM_ Left: 5 -CAGTTCCTCGTGCCCATC -3 Right: 5 -GGCAAAGAACGTGTTGACC U19720 Left: 5 -TACGAGTGCTCCCCCAACT -3 Right: 5 -CGTTCAGTACCCGCTCTTTG NM_ Left: 5 -CTCTGCTCCTCCTGTTCGAC-3 Catalog # ---- Right: 5 -GCCCAATACGACCAAATCC

5 Table 3S. Antitumor Efficacy Evaluation of Compound 1 and Taxol Against Early Stage Human HEPG2 in Female SCID Mice (under Folate Deficient and Standard Folate Replete Diet Conditions) Cg Treatment Drug Route 1 No Rx (Folate deficient) Schedule (start d4) Total Dosage mg/kg Drug Deaths 2 Compound 1 IV Q4dx /5 6 Taxol IV Q2dx6 45 0/5 Cg Treatment Drug Route 8 No Rx (STD diet) Schedule (Start on day 4) Total Dose mg/kg Drug Deaths 9 Compound 1 IV Q4dx /5 11 Taxol IV Q2dx6 45 0/5 Median Tumor Burden in mg on day 21 (range) 477 (0-752) 0 (0-875) 75 (0-234) Median Tumor Burden in mg on day 21 (range) 548 ( ) 546 ( ) 0 (0-88) T/C mass% Tumor Free on day 28 0/5 0 0/5 16 0/5 T/C mass% Tumor Free on day 31 0/5 99 0/5 0 0/5 Median Time to 500 mg in days (range) 22 (20-41) 35 (18-38) 29 (27-33) Median Time to 500 mg in days (range) 21 (20-31) 21 (18-22) 31 (30-39) Tumor Growth Delay (days) Log Kill Activity Rating Tumor Growth Delay (days) Log Kill Activity Rating NA None The NCR SCID mice were implanted bilaterally subcutaneously with 30-60mg tumor fragments by a 12-gauge trocar on day 0. Chemotherapy was started on day 4 after tumor implantation, when the number of cells was relatively small ( cells). A T/C= 0 indicates very high antitumor activity. The conversion of log tumor cell kill (LK) to activity rating is as follows: >2.8 LK = ++++ (highly active); LK = +++; LK = ++; LK = +; <0.7 = (inactive). + = positive + for LK but negative for T/C. Notes: The ranges reflect the actual tumor measurements for each mouse (e.g., right and left flank tumor (mg) values are added together). For cage 1, these values were 0, 88, 477, 515, and 752 mg for the untreated control. For cage 2, for the compound 1-treated mice, these values were 0, 0, 0, 126 and 875 mg. 5

6 A FR alpha R elative Tra nscr ipts TE-85 HTB166 MCF -7 MDA-MB231 MDA-MB435 T-47D KB HeLa HCT-116 SW-620 HCT 15 Caco-2 BCPC-3 UCV A O ACHN IGROV-1 OVCAR-3 SKOV-3 HepG2 Hep3B Y79 SK-MEL5 SK-MEL 28 HTB139 SK-N-SH SK-N-BE SK-N-MC HT1080 PC-3 DU-145 H1650 H2122 H2030 H1573 H1781 H3255 A549 CRL 5807 CRL5872 CRL5810 CRL5800 H596 NCI460 H69 H446 H226 H2373 H2452 H2461 H2591 H2595 H2714

7 B FR alpha Relative Transcripts U93 7 CTS Kasu mi- 1 HL 60 MV4-11 THP- 1 C AML- 193 KG-1 KG- 1a K562 CMS Meg-01 Dami CM K1 6 CMY 6 97 Nalm6 Uoc REH CCRF -CEM M OLT4 MOLT 3 HBP- AL L TALL- 1 DND41 ALL-SIL Jurkat PCFT Rel ati ve Tran script s U9 37 CT S Ka su mi -1 HL 60 M V4-1 1 T HP-1 AM L KG -1 KG -1a K5 62 CM S M eg-01 Da mi CM K16 CM Y 69 7 Nal m6 Uoc REH CCRF-CEM M OLT4 MO LT3 HBP-ALL TALL -1 DND41 ALL -SIL Jurk at D RFC Rela tiv e T ransc rip ts U93 7 CTS Ka sumi-1 HL 60 M V4-11 THP- 1 AM L- 193 KG- 1 KG -1a K5 62 C MS Me g- 01 Dami CM K16 CM Y 6 97 N alm6 Uoc REH CCRF- CEM M OL T 4 M OL T 3 HBP-ALL TA LL -1 DND4 1 AL L -SIL J urka t 7

8 Figure 1S. Transcripts for FRα, PCFT and RFC in solid tumor and leukemia cell lines. Transcripts were measured by real-time reverse transcription PCR (qrt-pcr) from total RNAs prepared from solid tumor cell lines (A) and leukemia cell lines (B-D) using a Roche480 Lightcycler and hydrolysis probes and primers specific for FRα (A and B) PCFT (C) and RFC (D). Transcript levels were normalized to GAPDH transcripts. Primer sequences and probes are listed in Table 2S. 8

9 A R1-11-PCFT4 Cpd 1 Cpd PG5 CPM PG 4 PG 3 PG % 14% 18% 8% 36% Fract ion number B 6500 HepG2 Pmx PG Pmx PG 6 PG 3 CPM % PG 4 PG % % 14% 10% 9% Fr action number C R1-11-PCFT4 Cpd 1 ± Conjugase Cp d 1 (+) Conjugase (-) Conjugase 600 PG 5 PG CPM 400 PG PG 3 78% % 22% 53% 100 9% 9% Fraction number Figure 2S. HPLC analysis of polyglutamyl derivatives of compound 1 and pemetrexed (Pmx) in R1-11-PCFT4 and HepG2 cells at ph 6.8. R1-11-PCFT4 (A and C) and HepG2 (B) cells were treated with 1 µm [ 3 H]compound 1 (A and C) or Pmx (B) at ph 6.8 in the presence of adenosine (60 µm) (compound 1) or adenosine (60 µm) and thymidine (10 µm) (Pmx) for 16 h. Polyglutamates were extracted by boiling in 50 mm sodium phosphate buffer (ph 6.0) containing 100 mm 2-mercaptoethanol and separated on a 5 µm Spherisorb C-18 ODS-2 column (4.6 mm x 250mm) with a Nova-Pak 4 µm C-18 guard column. Fractions were collected and measured for radioactivity. Treatment with chicken pancreas gamma glutamyl hydrolase ( conjugase ) (C) was used to validate the identity of the peaks. Details are described in Materials and Methods. 9

10 DMSO 0.5µM Cpd1 (µm) % SubG1 % G1 % S % G2 1.0µM DMSO µM Counts 10.0µM Propidiu m Iodide Figure 3S. Treatment of R1-11-PCFT4 cells with compound 1 induces S-phase accumulation and a modest level of apoptosis. The cell cycle profiles of R1-11-PCFT4 cells were examined by flow cytometry after 48 h of treatment with increasing concentrations (0.5, 1, 5, and 10 µm) of compound 1 at ph 6.8, compared to a control (DMSO). Cell cycle profiles of PI-stained cells and the percentages of SubG1 (apoptotic fraction) (white trace), G1 (dark grey trace), S (light grey trace), and G2 (grey trace) are tabulated and are presented graphically in Figure 7B. 10