US 20160243114A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0243114A1 Chen (43) Pub. Date: Aug. 25, 2016 (54) METHODS OF TREATING CANCERUSING Publication Classification COMPOSITIONS COMPRISING PERILLYL ALCOHOL DERVATIVE (51) Int. Cl. A613 L/495 (2006.01) (71) Applicant: NEONCTECHNOLOGIES INC., Los A63L/045 (2006.01) Angeles, CA (US) A6II 45/06 (2006.01) A647/48 (2006.01) (72) Inventor: Thomas Chen, La Canada, CA (US) (52) U.S. Cl. CPC ............. A6 IK3I/495 (2013.01); A61 K47/481 (21) Appl. No.: 15/026,649 (2013.01); A61 K3I/045 (2013.01); A61 K 45/06 (2013.01) (22) PCT Fled: Oct. 8, 2014 (57) ABSTRACT PCT NO.: PCT/US1.4/596OO (86) A method for treating brain metastases of a cancer in a mam S371 (c)(1), mal includes administering to the mammal atherapeutically (2) Date: Apr. 1, 2016 effective amount of a perillyl alcohol carbamate, such as TMZ-POH. The brain metastases can be originated or spread from breast cancer. The penllyl alcohol derivative may be Related U.S. Application Data perillyl alcohol conjugated with a therapeutic agent, Such as a chemotherapeutic agent. The chemotherapeutic agents that (63) Continuation of application No. 14/455.293, filed on may be used in the present invention include a DNA alkylat Aug. 8, 2014. ing agent, a topoisomerase inhibitor, an endoplasmic reticu (60) Provisional application No. 61/888.253, filed on Oct. lum stress inducing agent, a platinum compound, an antime 8, 2013. tabolite, an enzyme inhibitor, and a receptor antagonist. Patent Application Publication Aug. 25, 2016 Sheet 1 of 21 US 2016/0243114A1 cytotoxicity of DMC in glioma cells - 48h 100 8 axx U.S. 30 x (EE V 388x2 8 60 2 A s R 20 O 5. 50 O O Concentration uM) Figure 1 10 or cytotoxicity of POH-DMC in glioma cells-48h OO ex: 8.2 -0 O d 50 30 100 Concentration (uM) Figure 2 Patent Application Publication Aug. 25, 2016 Sheet 2 of 21 US 2016/0243114A1 Human glioma cells treated with TMZ for 48h 60 - 40 2-4-w, U-87 a-row A172 wrixo J251 O 50 100 150 200 120 Human glioma cells treated with TMZ-POH for 48h Concentration (uM) Figure 4 Patent Application Publication Aug. 25, 2016 Sheet 3 of 21 US 2016/0243114A1 MTT Assay for Rolipram and POH-Rolipram on glioma A172 cells - 48 h treatment 100 w--Rolipram 6.3. OO --POH-Rolipram 4 O ...w.g...w...w... P.--me wir O 2OO 400 600 300 OOO 100 Concentration (uM) Figure 5 MTT Assay for Rolipram and POH-Rolipram on glioma 120 . U-87 cels - 48 h treatment 100 -- Rolipram 30 ta was owPOH-Rolipram 60 " 8. 40 - 20 -200 O 200 400 600 800 OOO 1200 Concentration (uM) -------- Figure 6 Patent Application Publication Aug. 25, 2016 Sheet 4 of 21 US 2016/0243114A1 MTT Assay for Rolipram and POH-Rolipram on glioma U251 cells - 48h treatment re-Rofipram mir-POH-Rolipram OO O 2OO 400 600 800 1000 12OO Concentration (uM) Figure 7 MTT Assay for Rolipram and POH-Rolipram on glioma 12O N229 cells - 48 h treatment 100 mor-Rolipram : -I-POH-Rotipram 80 40 20 w -20) O 2OO 4OO 600 8OO OOO 1200 Concentration (uM) Figure 8 Patent Application Publication Aug. 25, 2016 Sheet 5 of 21 US 2016/0243114A1 Purified POH Figure 9A Patent Application Publication Aug. 25, 2016 Sheet 6 of 21 US 2016/0243114A1 Average Tumor Growth Over Time 3000 2500 so PBS Control --Purified POH 2 O O O -A-Sigma -x-Butyryl-POH 11 O5 OO OO 500 O Relegate 1 8 13 16 20 23 26 29 32 34 36 .38 40 42 44 46 48 50 53 55 57 59 Treatment Day Figure 9B Patent Application Publication Aug. 25, 2016 Sheet 7 of 21 US 2016/0243114A1 Colony Forming Assay for TMZ and TMZ-POH on TMZ sensitive and resistant glioma cells - 72 hr treatment 120 100s ---- - - {TMZ) 8 se Tr(TP) e is TZ 6 O O D MZ-POH Conjugate (TP) 40 N 2 u251 (TP) - U251 (TMZ) 10 2. 30 40 50 60 Drug (uM) Figure 10 CFA of U251 and U251TR with POH - 72 hr treatment. --O. U251 120 --O-- U25 TR 100 O 2. POH (mM) Figure 11 Patent Application Publication Aug. 25, 2016 Sheet 8 of 21 US 2016/0243114A1 MTT Assay for TMZ-POH on U251, U251TR, and normal Astrocytes-72 hr treatment. 2C-T- - -Q-0--- O----- -:i. -A w 60 stsY------ - 3 & N. --- - s -- | O 20 8 80 OO TMZ-POH (uM) Figure 12 MTT Assay for TMZ-POH on BEC, TUBEC, and Astrocytes - 72 hr treatment. O 20 40 60 8 100 TMZ-POH (uM) Figure 13 Patent Application Publication Aug. 25, 2016 Sheet 9 of 21 US 2016/0243114A1 MTT Assay for TMZ-POH on Glioma Cancer Stem Cell USC-04 - 72 hr treatment. --TMZ-POH -TMZ-- 1OO 20 3OO OO Drug Con'c(uM) Figure 14 MTT Assay for POH on Glioma Cancer Stem Cell USC-04-72 hr treatment. 16 40 2O 100 a. 8) --- 60 s S. - 40 - w 2O O -e-POH O.O O5 O 15 2.O. POH (mM) Figure 15 Patent Application Publication Aug. 25, 2016 Sheet 10 of 21 US 2016/0243114A1 MTT Assay for TMZ-POH on Glioma Cancer Stem Cell USC-02 72 hr treater. 12 O 40 > - TMZ-POH |- TMZ-POH --- O --TMZ *r. O 50 1 OO 50 2OO Drug Con'c (uM) Figure 16 MTT Assay for POH on Glioma Cancer Stem Cell USC-02-72 hr treatent. 40 8 O MO) ' 0 %troxwww.gxw-oxwoxx x O. O.5. O 5 POH (mM) Figure 17 Patent Application Publication Aug. 25, 2016 Sheet 11 of 21 US 2016/0243114A1 Western Blot following TMZ-POH treatment of U251 TMZS and U251-TMZr Cells - 18 hr treatment O 20 40 60 80 100 150 200 TMZ-POHuM) O 20 40 60 80 100 150 200 PARP an aii is one in ele PARP CHOP ACTIN U251-TMZS U251-TMZr Figure 18 Patent Application Publication Aug. 25, 2016 Sheet 12 of 21 US 2016/0243114A1 : | 100 200 300 400 500 C O d L O S 2 s u) 0.001-, w w O 20 40 60 80 1 OO O 250 5OO 750 1000 Concentration uM) Concentration uM Figure 19 Patent Application Publication Aug. 25, 2016 Sheet 13 of 21 US 2016/0243114A1 : 20 30 40 Concentration uM) Figure 20A X Š Š S. S š. & Figure 20B Patent Application Publication Aug. 25, 2016 Sheet 14 of 21 US 2016/0243114A1 Figure 21A MGMT Actin Figure 21B - T-P TMZ O6-BG vh. O 30 5050 20010 500 (uM) MGMT ACtin Figure 21C Patent Application Publication Aug. 25, 2016 Sheet 15 of 21 US 2016/0243114A1 Figure 22A O 1. 231-MGMT-1 231-MGMT-2 : w O.O1 x 2OO 3OO 4OO O 1OO 200 300 400 Concentration uM Concentration uM Figure 22B Patent Application Publication Aug. 25, 2016 Sheet 16 of 21 US 2016/0243114A1 Figure 23A 231 MGMT-1 ecoc<c) ©CDO Figure 23B == --No. MDAMB-468 Figure 23C Patent Application Publication Aug. 25, 2016 Sheet 17 of 21 US 2016/0243114A1 Actin Figure 24A Figure 24B TMZ'Y' poh POH Š: Y-H2AX Figure 24C Actin Figure24D Patent Application Publication Aug. 25, 2016 Sheet 18 of 21 US 2016/0243114A1 C. PARP Actin Figure 25B Patent Application Publication Aug. 25, 2016 Sheet 19 of 21 US 2016/0243114A1 125 3. 1OO S 75 O o 50 O 25 - is 8 S 8 Patent Application Publication Aug. 25, 2016 Sheet 20 of 21 US 2016/0243114A1 Day 43 X107 Figure 27A Patent Application Publication Aug. 25, 2016 Sheet 21 of 21 US 2016/0243114A1 Sssssssss days after tumor implantation Figure 27B US 2016/02431 14 A1 Aug. 25, 2016 METHODS OF TREATING CANCERUSING 0005 Metastasized cancer, such as breast cancer, that has COMPOSITIONS COMPRISING PERILLYL spread to the brain poses a similarly serious therapeutic chal ALCOHOL DERVATIVE lenge as malignant gliomas. This challenge once was a late aspect of disease progression, but increasingly is becoming a CROSS REFERENCE TO RELATED first site of disease progression after otherwise Successful APPLICATIONS treatment of primary tumor and metastases outside the cra nium. Traditional breast cancer therapeutics, such as pacli 0001. This application claims priority to U.S. Provisional taxel or doxorubicin, only reach brain metastases at concen Application No. 61/888.253, filed Oct. 8, 2013. This applica trations that are far lower than needed to be therapeutically tion also relates to U.S. application Ser. No. 14/455.293 filed active. P. R. Lockman, et al. Heterogeneous blood-tumor Aug. 8, 2014, which is a continuation of U.S. application Ser. barrier permeability determines drug efficacy inexperimental No. 13/566,731 filed Aug. 3, 2012, which is a continuation of brain metastases of breast cancer, Clin Cancer Res 16 (2010) International Application No. PCT/US2011/049392 filed 5664-5678. The most critical barrier to effective entry of Aug. 26, 2011, which claims priority to U.S. Provisional chemotherapeutics into the brain is the blood brain barrier Application Nos. 61/377,747 (filed Aug. 27, 2010) and (BBB), and very few anticancer drugs are able to overcome 61/471.402 (filed Apr. 4, 2011). The disclosures of all of these this obstacle. E. Fokas, J. P. Steinbach, C. Rodel, Biology of prior filed applications are incorporated by reference herein brain metastases and novel targeted therapies: time to trans in their entireties.