VAKILI MOHAMMAD REZA (CA)
SADAT SAMS MOHAMMAD ANOWAR (CA)
WO2018184113A1 | 2018-10-11 | |||
WO2015023551A1 | 2015-02-19 |
O'DWYER PETER J , CATALANO ROBERT B: "Uridine diphosphate glucuronosyltransferase (UGT) 1A1 and irinotecan: practical pharmacogenomics arrives in cancer therapy.", JOURNAL OF CLINICAL ONCOLOGY, AMERICAN SOCIETY OF CLINICAL ONCOLOGY, US, vol. 24, no. 28, 1 October 2006 (2006-10-01), US , pages 4534 - 4538, XP002665591, ISSN: 0732-183X, DOI: 10.1200/JCO.2006.07.3031
DE PAIVA IGOR MOURA, VAKILI MOHAMMAD REZA, SOLEIMANI AMIR HASAN, TABATABAEI DAKHILI SEYED AMIRHOSSEIN, MUNIRA SIRAZUM, PALADINO MA: "Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis", MOLECULAR PHARMACEUTICS, AMERICAN CHEMICAL SOCIETY, US, vol. 19, no. 6, 6 June 2022 (2022-06-06), US , pages 1825 - 1838, XP093050637, ISSN: 1543-8384, DOI: 10.1021/acs.molpharmaceut.1c00918
SADAT SAMS M. A., WUEST MELINDA, PAIVA IGOR M., MUNIRA SIRAZUM, SARRAMI NASIM, SANAEE FORUGHALSADAT, YANG XIAOYAN, PALADINO MARCO,: "Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage", FRONTIERS IN ONCOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 11, 23 December 2021 (2021-12-23), CH , pages 772920 - 17, XP093050639, ISSN: 2234-943X, DOI: 10.3389/fonc.2021.772920
WHAT IS CLAIMED IS: 1. A method of treating a subject having colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer, comprising: administering to said subject, - a therapeutically effective amount of A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, and - a therapeutically effective amount of SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 2. The method of claim 1, wherein the SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof is administered before administration of the A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 3. The method of claim 1 or 2, wherein the A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, , or prodrug thereof is administered before administration of the PM16-SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 4. The method of claim 1, wherein the SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof is administered substantially concurrently with the A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 5. The method of any one of claims 1 to 4, wherein the subject is a human. 6. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of A83B4C63 is administered in a micelle. 7. The method of claim 6, wherein the A83B4C63-containing micelle comprises block copolymer mPEO-b-PBCL, PEO-b-PBCL, PEO-PCL, PEO-PDLA, and/or PEO-PLGA. 8. The method of claim 7, wherein the A83B4C63-containing micelle comprises block copolymer mPEO-b-PBCL. 9. The method of claim 8, wherein the PBCL block of the block copolymer in the A83B4C63-containing micelle has a degree of polymerization of between 2 and 50, preferably 10-30. 10. The method of claim 9, wherein the PBCL block of the block copolymer in the A83B4C63-containing micelle has a degree of polymerization of 26. 11. The method of claims 6-10, wherein the concentration of A83B4C63 in the A83- containing micelle is between 1 µM and 100 µM, preferably between 5 µM and 40 µM. 12. The methods of any one of claims 1 to 11, wherein the therapeutically effective amount of SN-38 is administered in a micelle. 13. The method of claim 12, wherein the SN-38-containing micelle comprises block copolymer mPEO-b-PBCL, PEO-b-PBCL, PEO-PCL, PEO-PDLA, and/or PEO-PLGA. 14. The method of claim 13, wherein the SN-38-containing micelle comprises block copolymer mPEO-B-PBCL. 15. The method of claims 13 to 14, wherein SN-38 in the SN-38-containing micelle is conjugated to a block copolymer. 16. The method of claims 13 to 15, wherein the PBCL block of the block copolymer in the SN-38-containing micelle has a degree of polymerization of between 1 and 30, preferably between 5 and 25. 17. The method of claims 13 to 16, wherein the PBCL block of the block copolymer in the SN-38-containing micelle has a degree of polymerization of 16. 18. The method of claims 12 to 17, wherein the concentration of SN-38 in the micelle is between 0.001 µM and 10 µM, preferably between 0.005 µM and 1 µM. 19. The method of claim 1 to 5, wherein the therapeutically effective amount of A83B4C63 or SN-38 is administered in a composition. 20. The method of claim 1 to 5, wherein the therapeutically effective amount of A83B4C63 or SN-38 is administered in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, diluent, or carrier. 21. Use of a therapeutically effective amount of A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, and a therapeutically effective amount of SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, for treating a subject having colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer . 22. Use of a therapeutically effective amount of A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, and a therapeutically effective amount of SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, in the manufacture of a medicament for treating a subject having colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer. 23. The use of claim 21 or 22, wherein the SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof is for administration before administration of the A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 24. The use of claim 21 or 22, wherein the A83B4C63, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof is for administration before administration of the SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 25. The use of claim 21 or 22, wherein the SN-38, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof is for administration substantially concurrently with the A83, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. 26. The use of any one of claims 18-25, wherein the subject is a human. 27. A micelle composition comprising SN-38 and A83B4C63. 28. The micelle composition of claim 24, wherein the micelle comprises block copolymer mPEO-b-PBCL, PEO-b-PBCL, PEO-PCL, PEO-PDLA, and/or PEO-PLGA. 29. The micelle composition of claim 28, wherein the micelle comprises block copolymer mPEO-b-PBCL. 30. The micelle composition of claims 27 to 29, wherein SN-38 is conjugated to a block copolymer. 31. The micelle composition of claims 27 to 30, wherein the PBCL block of the block copolymer has a degree of polymerization between 1 and 50. 32. The micelle composition of claim 27 to 31, wherein the concentration of A83B4C63 is between 1 µM and 100 µM, preferably between 5 µM and 40 µM, and the concentration of SN-38 is between is between 0.001 µM and 10 µM, preferably between 0.005 µM and 1 µM. 33. A composition comprising SN-38 and A83B4C63. 34. A pharmaceutical composition comprising SN-38 and A83B4C63, a pharmaceutically acceptable excipient, diluent, or carrier. 35. A method of treating colorectal cancer in a subject with of treating colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer, comprising: administering to said subject a therapeutically effective amount a micelle composition of claims 27 to 32, a composition of claim 33, or a pharmaceutical composition of claim 34. 36. The method of claim 35, wherein the subject is a human. 37. Use of a therapeutically effective amount of a micelle composition of claims 27 to 32, a composition of claim 33, or a pharmaceutical composition of claim 34, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, for treating a subject having colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer. 38. Use of a therapeutically effective amount of a micelle composition of claims 27 to 32, a composition of claim 33, or a pharmaceutical composition of claim 34, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, in the manufacture of a medicament for treating a subject having colorectal cancer, suspected of having of treating colorectal cancer, or at risk of developing of treating colorectal cancer. 39. The use of claim 37 or 38, wherein the subject is a human. |
[00119] 2.7. Transmission electron microscopy (TEM) [00120] The morphology of self-assembled structures under study was investigated by TEM using a Morgagni TEM (Field Emission Inc., Hillsboro, OR) with Gatan digital camera (Gatan, Pleasanton, CA). In brief, 20 μL of micellar solution with a polymer concentration of 0.5 mg/mL was placed on a copper-coated grid. The grid was held horizontally for 1 min to allow the colloidal particles to settle down. The excess fluid was removed by filter paper. The copper-coated grids holding the aqueous samples were then negatively stained by 2% phosphotungstic acid. After 2 min, the excess fluid was removed by filter paper and the grid was loaded into the TEM for image analysis. [00121] 2.8. In Vitro drug release [00122] In vitro release of SN-38 and A83B4C63 from the self-assembled structures was investigated using dialysis-bag diffusion technique. Each dialysis bag (Spectra Por dialysis tubing, MWCO = 3.5 kDa, Spectrum Laboratories, Rancho Dominguez, CA, U.S.A.), containing 3 mL of the micellar formulation in water or free SN- 38 and A83B4C63 dissolved in DMSO, was immersed into 300 mL release medium (4% albumin in ultrapure water) maintained at 37ºC in a shaking water bath with 65 rpm (Julabo SW 22, Seelbach, Germany). At selected time intervals (0, 1, 2, 4, 6, 8, and 24 h), 300 μL aliquots from inside of the dialysis bag were withdrawn and replaced with equal volume of fresh release media (water). The concentrations of SN-38 in collected samples were determined by UV-Vis spectrophotometer (BioTEK, Winooski, VT, USA). Detection was performed at a wavelength of 383 nm. The concentrations of A83B4C63 in collected samples were measured and analyzed using a Varian Prostar 210 HPLC system. All experiments were carried out in triplicate. [00123] 2.9. Cell lines [00124] Two CRC cell lines, HCT116 and HT-29 originally purchased from ATCC were used. The cells were cultured at 37ºC in 5% CO 2 in a humidified incubator in a 1:1 mixture of Dulbecco’s modified Eagle medium and F12 (DMEM/F12) supplemented with 10% FBS, 50 U/mL penicillin, 50 mg/mL streptomycin, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate. All culture supplements were purchased from GIBCO, Life Technologies Inc. (Burlington, ON, CA). [00125] 2.10. In vitro synergy and antagonism evaluation [00126] The day before the treatment, 2 × 10 3 HCT116 and HT-29 cells were plated in each well of 96-well flat-bottomed plates. Then cells were treated with irinotecan, PM 26 /A83, SN-38, PM 16 -SN-38, irinotecan + PM 26 /A83, SN-38 + PM 26 /A83, PM 16 -SN-38 + PM 26 /A83 for 48 h. The cells were treated with the formulations at 0.001, 0.01, 0.1, 1, 10, and 100 µM concentrations for irinotecan and SN-38 formulations. The treating concentrations of A83B4C63 as part of formulations were 5, 10, 20, and 40 µM for the combination treatments and the control cells received only 0.1% DMSO. After the incubation time point, cells were treated with the MTS reagent (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega, USA) to measure their viability. Each experiment was performed in quadruplicates. The cell viability percentages were calculated using the following formula: [00127] The cell viability data following a combination template was processed using Loewe classical synergy models in Combenefit ® software program [15]. [00128] 2.11. In vitro cytotoxicity assay [00129] The CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) kit from Promega, USA was used to assay the cytotoxicity of irinotecan, irinotecan + PM 26 /A83, SN-38, SN-38 + PM 26 /A83, PM 16 -SN-38, PM 16 -SN-38 + PM 26 /A83, PM 16 -SN- 38:PM 26 , and PM 16 -SN-38:PM 26 /A83 in HCT116 and HT-29 cells according to the provided company protocol. In brief, 2 × 10 3 cells were plated in each well of 96-well flat- bottomed plates 24 h prior to the treatments. Then, cells were treated with the formulations at the concentration range of 0.001 to 100 µM for irinotecan and SN-38 formulations. The PM 26 /A83 with a fixed concentration as of 10 µM was used for the combination treatments and the control cells received only 0.1% DMSO. The choice of this concentration was based on the results of synergy evaluation, in which at a concentration of 10 µM for A83B4C63, a synergy with SN-38 at a concentration range of 0.01-1 µM has been observed. After different experimental incubation time points (24, 48, and 72 h), 20 µL of MTS reagent was added in each well and further incubated for 1 h at 37ºC before measuring the absorbance at 490 nm using BioTEK microplate reader. Each experiment was performed in quadruplicates. The cell viability percentages were calculated using the formula mentioned in previous method section. [00130] 2.12. Western blot analysis [00131] Western blotting was performed to assess the in vitro levels of caspase-3, caspase-7, PARP, and γ-H2AX proteins after treating both HCT116 and HT-29 cell lines with A83, irinotecan, irinotecan + PM 26 /A83, SN-38, SN-38 + PM 26 /A83, PM 16 -SN-38, PM 16 -SN-38 + PM 26 /A83, PM 16 -SN-38:PM 26 , PM 16 -SN-38:PM 26 /A8B4C63, and PM 26 /A83. In brief, 1 × 10 6 cells were plated in each well of 6-well plates 24 h prior to the treatments. Then, the cells were treated with the media containing various treatments the final concentrations equivalent to the respective relative IC 50 concentrations of irinotecan, SN- 38, PM 16 -SN-38, and PM 16 -SN-38:PM 26 for 6 h in combination with or without 10 µM PM 26 /A83. The cells of 3 controls received only 0.1% DMSO, 10 µM A83, and 10 µM PM 26 /A83, respectively. Protein extracts for western blot analysis were prepared using commercial RIPA lysis buffer (ThermoFisher Scientific, Canada) supplemented with a cocktail of protease inhibitors (Millipore Sigma, Canada). Protein concentrations were measured using the BCA assay kit (Pierce/ThermoFisher Scientific, Canada) according to the manufacturer’s protocol. Equal concentrations of protein were separated by SDS- PAGE and transferred to nitrocellulose membranes. After blocking with 5% skimmed milk in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20), the blots were incubated with the respective primary antibodies (cleaved caspase-3 cat# 9661S, cleaved caspase-7 cat# 9491S, PARP cat# 9542S, phospho-histone H2A.X (Ser139) cat# 9718S) and secondary antibody (HRP-linked anti-rabbit IgG cat# 7074S) purchased from Cell Signaling Technology (Whitby, ON, Canada). The protein bands were detected using an enhanced chemiluminescence (ECL) based system (Pierce/ThermoFisher Scientific, Canada). The band intensities for the PNKP protein were quantified by performing optical density analysis using ImageJ software. Each experiment was carried out in triplicate. [00132] 2.13. Statistical analysis [00133] Data are shown as mean ± standard error of at least three experiments. GraphPad Prism9 software (La Jolla, CA, USA) was used for statistical analysis. The significance of difference between groups was assessed using one-way ANOVA followed by Tukey’s post-hoc analysis. If a significant difference was found among the groups, median ranks between pairs of groups were compared using the Mann-Whitney U test. Differences in the physicochemical characterization of micellar formulations were also tested using the unpaired student’s t-test. A value of p ≤ 0.05 was considered as statistically significant in all experiments. [00134] 3. Results [00135] 3.1. Characterization of block copolymers and PM formulations [00136] The 1 H NMR spectra of PM 16 -SN-38 and peak assignments are shown in Supplementary Fig.1. Using 1 H NMR data as reported before, the DP of PBCL block in mPEO-b-PBCL copolymers used for SN-38 conjugation or A83B4C63 loading was found to be 16 and 26, respectively [2, 11, 13, 16, 17]. The end-capped carboxylic acid functional group conversion of PBCL block of mPEO-b-PBCL copolymer was 65-67%. The conjugated content (% w/w) of SN-38 to mPEO-b-PBCL-COOH was 15.67 ± 0.34 as measured by UV spectroscopy. [00137] The physicochemical characterizations of the self-assembled PM 16 -SN-38, PM 16 -SN-38:PM 26 , PM 16 -SN-38:PM 26 /A83, PM 26 /A83, and PM 26 micelles were also performed by measuring their average diameter, surface charge, PDI, and CMC as summarized in Table 2. The average diameters of all the micellar formulations were below 100 nm with PDI range below 0.22. The average hydrodynamic diameter of PM 16 - SN-38 was significantly lower (p < 0.0001) than that of it mixed micelle with PM 26 , i.e., PM 16 -SN-38:PM 26 (without A83). Co-encapsulation of PM 16 -SN-38 and A83B4C63 in PM 16 -SN-38:PM 26 /A83 resulted in larger size (58.41 ± 0.29 nm) among all the formulations under study. [00138] The mean zeta potential for PM 16 -SN-38 was on the negative side. The zeta potential of PM 26 /A83 and PM 26 , on the other hand was neutral. When PM 16 -SN-38 formed mixed micelles with PM 26 (without or with A83B4C63 encapsulation), i.e., in PM 16 - SN-38:PM 26 , PM 16 -SN-38:PM 26 /A83, the zeta potential shifted towards the neutral range (from 0.07 to 0.08 mV), which was significantly different from the zeta potential of PM 16 - SN-38 micelles (p < 0.003) but not different from that of PM 26 samples. [00139] The measured CMCs of PM 16 -SN-38, PM 16 -SN-38:PM 26 , PM 16 -SN- 38:PM 26 /A83, PM 26 /A83, and PM 26 were 0.33 ± 0.09, 0.54 ± 0.12, 0.47 ± 0.22, 0.43 ± 0.11, and 0.48 ± 0.21 µg/mL, respectively (Table 2). No statistical significance was identified between the CMCs of the formulations under study. [00140] The loading of SN-38 in the mixed micellar formulation, i.e., PM 16 -SN- 38:PM 26 , PM 16 -SN-38:PM 26 /A83, of that in the PM 16 -SN-38 owing to 1:1 molar mixing ratio of polymers. The A83B4C63 encapsulation, was found to significantly decrease in mixed micellar formulation of PM 16 -SN-38:PM 26 /A83 co-encapsulating both SN-38 and A83B4C63, compared to that of PM 26 /A83 (p < 0.05) (Table 2). [00141] 3.2. Transmission electron microscopy (TEM) [00142] The morphology of the self-assembled structures was investigated by TEM, confirming the formation of spherical-shaped PM 16 -SN-38, PM 16 -SN-38:PM 26 , PM 16 - SN-38:PM 26 /A83, PM 26 /A83, and PM 26 micelles with uniform size (Fig.2A-2E). Moreover, a similar distribution pattern in the micellar population having a clear boundary was observed in TEM images of all micelles, indicating the lower aggregation tendency of micelles. Dynamic light scattering profiles showed formulation of a single peak for micelles formed from one or a mixture of block copolymers understudy (Fig 2F-2J). The trend of change in the average diameter and size distribution between samples was similar in the two methods of analysis, i.e., TEM and DLS. For instance, PM 16 -SN-38 showed the smallest average diameter and narrow polydispersity in both methods (Fig 2A and 2F). Whereas PM 16 -SN-38:PM 26 /A83 showed larger average diameter and high polydispersity (Fig 2C and 2H). [00143] 3.3. Kinetic stability [00144] The kinetic stability of block copolymer micelles was investigated by following the dissociation of micellar peak in the presence of a destabilizing surfactant, i.e., SDS, after 0, 1, 2, 4, 8, and 24 h incubation using DLS technique. Fig.3A and 3B represent the % intensity of micellar peak and PDI, respectively, for PM 16 -SN-38, PM 16 - SN-38:PM 26 , PM 16 -SN-38:PM 26 /A83, and PM 26 /A83 micelles over 24 h in the presence of SDS. All the micellar formulations exhibited complete resistance against the destabilizing agent and remained intact throughout the incubation period up to 24 h. As shown in Fig. 3B, no significant dissociation of the micellar structures was detected since the DLS- obtained PDI was in similar ranges (0.1 to 0.14) for the formulations in this study following incubation with SDS. Overall data suggest that all the micellar formulations are kinetically stable regardless of the payloads and their composition. [00145] 3.4. In vitro drug release [00146] Fig.4 shows the comparative in vitro release profiles of A83B4C63 and SN-38 from their respective formulations compared to free A83B4C63 and SN-38, under experimental conditions. Within 6 h, 98.81 ± 2.09% of A83B4C63 and 96.46 ± 1.43% of SN-38 as free drugs solubilize with the aid of DMSO were released from the dialysis bag indicating the existence of near sink condition [18]. In contrast, only 27.46 ± 2.53% and 43.88 ± 4.18% of A83B4C63 were released over 6 h from PM 26 /A83 and PM 16 -SN- 38:PM 26 /A83 micelles, respectively. Similarly, only 34.49 ± 5.23%, 28.28 ± 2.62%, and 20.70 ± 4.52% of SN-38 were released over 6 h from PM 16 -SN-38, PM 16 -SN-38:PM 26 , and PM 16 -SN-38:PM 26 /A83 micelles, respectively. In addition, the release of A83B4C63 from PM 16 -SN-38:PM 26 /A83 micelles over 24 h was 76.36 ± 1.41% (Fig.4A), which was significantly higher than that of PM 26 /A83 (42.70 ± 0.72) micelles and significantly lower that of free SN-38 (99.81 ± 0.01%). In contrast, the release of SN-38 from PM 16 -SN- 38:PM 26 /A83 micelles over 24 h was 44.15 ± 2.61%, which was significantly lower than that of PM 16 -SN-38 (74.16 ± 3.65) and PM 16 -SN-38:PM 26 micelles (57.94 ± 3.69%) (Fig. 4B). The overall in vitro release study supports the efficiency of micellar formulations for sustaining the release of both A83B4C63 and SN-38. It also showed release of SN-38 to slow down but release of A83B4C63 to increase upon formation of mixed micelles (PM 16 - SN-38:PM 26 /A83) compared to individual micelles encapsulating each drug alone (PM 16 - SN-38 or PM 26 /A83). [00147] 3.5. Evaluation of synergy between the combination treatments [00148] To further assess the synergistic versus additive effects of combinations of PNKP inhibitor and TOP1 inhibitor, and to predict the effective synergistic doses of these two drugs as combination therapy against CRC cell lines, we conducted software program-based analysis of our cell viability data. Fig.5 shows the graphical outputs from the Combenefit ® software that represent the combination dose response surface mapping the synergy as well as antagonism distribution. For interpreting the value of synergy scores, Combenefit ® software has normalized input data as cell viability inhibition percentage to directly infer the proportion of cellular responses attributing to the interactions of various concentrations of both drugs. Therefore, the combination scores near zero in the surface plot yields inadequate confidence on synergy or antagonism. However, the combination scores below -10, between -10 to 10, and above 10 for the combination of two drugs are likely to be considered antagonism, additivity, and synergy, respectively. Irinotecan in combination with PNKP inhibitor mostly showed additive scores, also confirming the inefficacy to kill CRC cell lines as revealed by the cell viability experiments at studied concentrations (Fig 5A and 5B). Loewe models suggested the clear synergistic effects for combining encapsulated PNKP inhibitor and TOP1 inhibitor SN-38 (as free or encapsulated) at a concentration range between 0.01 to 10 µM for TOP1 inhibitor and between 10 to 40 µM for PNKP inhibitor. Since both SN-38 and PM 16 - SN-38 are extremely toxic to cells, the combinatorial effects were not considered synergistic at higher concentrations of SN-38 by the software. When combining free SN- 38 with PM 26 /A83, the synergy scores reached to 50 in 10-40 µM range of A83B4C63 concentration (Fig.5C and 5D). Combining PM 26 /A83 with PM 16 -SN-38 produced a synergy score of 50 between 10 and 40 µM concentrations for A83B4C63 and 0.01 and 1 µM concentrations for SN-38 (Fig.5E and 5F). [00149] 3.6. In vitro cytotoxicity [00150] The MTS assay was performed at 24 - 72 h to determine the anticancer activity for the combination of TOP1 and PNKP inhibitors with or without nano-delivery against CRC cell lines (HCT116 and HT-29) in comparison to empty polymeric micelles, TOP1 inhibitors SN-38 or irinotecan alone, and PNKP inhibitor alone (Fig.6). [00151] As summarized in Table 3, in HCT116 the relative IC 50 of irinotecan was 7.3 ± 3.7, 3.9 ± 1.7 and 6.3 ± 1.6 µM following 24, 48, and 72 h incubation. Upon combination of irinotecan with PM 26 /A83 the relative IC 50 decreased to 5.7 ± 2.5, 4.2 ± 1.2, and 3.9 ± 1.0 µM at the same incubation times, respectively. The difference in mono versus combination therapy was significant for the 72 h incubation time. Similar trend was observed in HT-29 cell line, where the decrease in relative IC 50 between mono and combination therapy was found to be significant after 48 h incubation time. As expected, at similar concentration range, treatment of both cell lines with SN-38 led to higher cytotoxicity compared to that for irinotecan (Table 3). In both cell lines, PM 16 -SN-38 was shown to have similar relative IC 50 to that of free SN-38, in all incubation times. [00152] Adding PM 26 /A83 to both cell lines while treating them with either free SN- 38 or PM 16 -SN-38 led to a decrease in the relative IC 50 of SN-38 in all incubation times and in both cell lines. For instance, in HCT116 cells, the relative IC 50 of free SN-38 was decreased from 0.14 ± 0.04, 0.05 ± 0.01, 0.02 ± 0.01 µM at 24, 48, and 72 h to 0.012 ± 0.001, 0.007 ± 0.001, and 0.002 ± 0.001 µM upon combination with PM 26 /A83. For PM 16 - SN-38, co-treatment of HCT116 cells led to a decrease to relative IC 50 from 0.12 ± 0.05, 0.05 ± 0.01, and 0.01 ± 0.002 µM to 0.013 ± 0.001, 0.008 ± 0.001, and 0.003 ± 0.001 µM for 24, 48 and 72 h incubation times, respectively (*p < 0.05, student’s t-test). The results showed similarly superior cytotoxicity of SN-38 + PM 26 /A83 and that of PM 16 -SN- 38+PM 26 /A83 samples over other treatments at all incubation time points against both cell lines. [00153] We then made a comparison between the cytotoxicity of PM 16 -SN-38:PM 26 and that of PM 16 -SN-38:PM 26 /A83 co-delivery system (Fig.7 and Table 4). Similar to combination treatment of PM 16 -SN-38 + PM 26 /A83 that showed higher cytotoxicity in HCT116 and HT-29 cell lines compared to PM 16 -SN-38, alone; the co-delivery system of PM 16 -SN-38:PM 26 /A83 was more cytotoxic than PM 16 -SN-38:PM 26 . In the HCT116 cells, the average relative IC 50 of co-delivery system was 0.02, 0.03 and 0.01 µM at 24, 48, and 72 h incubation, based on SN-38 concentration, while PM 16 -SN-38:PM 26 without A83, showed relative IC 50 s of 0.19, 0.16, and 0.03 µM at the same incubation times. This represents a 9.5, 5.3, and 3 fold decrease in the relative IC 50 of the co-delivery system over the same polymeric micellar structure without A83, which was similar to the level of reduction in relative IC 50 for the combination treatment of PM 16 -SN-38 + PM 26 /A83 compared to PM 16 -SN-38 alone. Similar trend was observed in HT-29 cells [00154] Table 4: Relative IC 50 range of PM 16 -SN-38:PM 26 and PM 16 -SN- 38:PM 26 /A83 against HCT116 and HT-29 cell lines after 24, 48, and 72 h of incubation (n = 4). The relative IC 50 values were determined after plotting the cell viability percentages vs. various drug concentrations using GraphPad Prism 9 software. The graph was then fitted with a non-linear regression and sigmoid dose-response curve to obtain the relative IC 50 values. The number shown in the subscript of the formulation names indicates the degree of polymerization of each block of the copolymers as determined by 1 H NMR spectroscopy [00155] 3.7. Expression of apoptosis mediators for the combination versus monotherapies [00156] As shown in Fig.8, PM 26, free A83 and PM 26 /A83 showed no apoptotic signaling reflecting the inertness and nontoxic characteristics of the delivery system and lack of off target activity by the PNKP inhibitor A83B4C63 at 10 µM. Addition of PM 26 /A83 as PNKP inhibitor to irinotecan significantly increased the expression of cleaved PARP, g- H2AX, cleaved caspase 3 and 7 in both cell lines. Addition of PM 26 /A83 to PM 16 -SN-38, did not affect cleaved PARP, g-H2AX expression significantly except in HT-29 cells where g-H2AX expression was increased with the co-treatment. The cleaved caspase 3 expression, however showed a significant increase in expression in both cell lines upon co-treatment with these formulations compared to monotherapy with PM 16 -SN-38, however (*p < 0.05). [00157] In HCT116 cells, the formulation designed for co-delivery of TOP1 and PNKP inhibitor, i.e., PM 16 -SN-38:PM 26 /A83 at its relative IC 50 concentration revealed significantly higher expression of cleaved PARP (*p ≤ 0.05, student’s t-test), when compared to the co-treatment with individual formulations, i.e., PM 26 /A83 + PM 16 -SN-38. However, no significant difference was observed for the expression of cleaved PARP between these treatments in HT-29 cell line. Moreover, in HT-29 cells, the PM 16 -SN- 38:PM 26 /A83 treatment showed significantly higher expression of g-H2AX (*p ≤ 0.05, student’s t-test) compared to the co-treatment with the individual formulations, i.e., PM 26 /A83 + PM 16 -SN-38. In contrast, no significant difference was observed for the expression of g-H2AX between these treatments in HCT116 cell line. Notably, no significant differences were obtained between co-delivery formulation (PM 16 -SN- 38:PM 26 /A83) and combination (PM 16 -SN-38 + PM 26 /A83) treatments in both cell lines in the expression cleaved caspase 3 and 7. Based on these data, the similar expression levels of these apoptosis mediators in CRC cell lines upon co-delivery (PM 16 -SN- 38:PM26/A83) and combination (PM16-SN-38 + PM26/A83) treatments demonstrated both structural integrity and anticancer efficacy of the payloads using polymeric micellar NPs. [00158] 4. Discussion [00159] The resistances to the conventional chemotherapies and the development of metastasis are the main causes of poor prognosis for advanced CRC. In last 2-3 decades, CRC patients have been exclusively treated with the chemotherapies based on fluoropyrimidines until the recognition of TOP1 enzyme as a valid therapeutic target in the cancer. As a result, a second generation semisynthetic camptothecin derivative, i.e., irinotecan as TOP1 inhibitor has been approved for clinical use in CRC patients. However, irinotecan is a prodrug which needs enzymatic conversion to produce its active metabolite, SN-38 in liver. Such conversion in liver needs higher frequent doses of irinotecan and eventually it results in suboptimal therapeutic responses, which requires combination treatment(s) in most advanced and some primary CRC patients [19]. [00160] Our research group has previously reported on the development of novel inhibitors of a DNA repair enzyme known as PNKP that can sensitize CRC to the cytotoxic effect of TOP1 inhibitors. We have also developed polymeric micellar formulations for SN-38 as well as a lead PNKP inhibitor known as A83B4C63 [2, 11, 20]. The primary objective of this study was to investigate the synergy between polymeric micellar formulations of SN-38 and A83B4C63 combination as individual micelles added together or mixed micelles co-delivering both drugs (Table 1) against CRC cell lines, in vitro. [00161] First, SN-38 was chemically conjugated to the poly(ester) end of mPEO-b- PBCL with a lower DP of 16, using activation of the COOH group at the polymer end. This has led to > 600 folds increase in water solubilized levels of SN-38 (25 µg/mL to over 15 mg/mL). The level of SN-38 conjugation to the carboxyl group at the PBCL end increased by ~5% where activation of carboxyl group by oxalyl chloride was pursued, compared to our previously reported SN-38 conjugation [11]. For comparison, the conjugated level of SN-38 to PEO-poly(glutamic acid) polymers in the NK012 formulation is reported at 20 % w/w, while SN-38 conjugation to the mPEO-b-PBCL end of this paper is at 16 % w/w [21]. [00162] With respect to A83B4C63 encapsulation, the use of mixed block copolymers of PM 16 -SN-38:PM 26 instead of PM 26 alone led to a decrease in the encapsulated levels of A83B4C63 (Table 2). Although the achieved loading still increased the water-soluble levels of A83B4C63, from 1 µM to 10.67. [00163] Micelles co-encapsulating SN-38 and A83, showed increased average diameter compared to polymeric micelles encapsulating each drug, although the average diameter of the co-delivery systems. was still < 60 nm (Table 2 and Fig.2). Like polymeric micelles delivering individual SN-38 or A83, the mixed micelles containing both drugs showed high thermodynamic and kinetic stability (Table 2 and Fig.3). The high stability of the polymeric micellar structure is mainly attributed to the presence of benzyl carboxylate group that may lead to p-p stacking and rigidity of the core structure [11]. Similar to micelles for individual drugs, the releases of SN-38 and A83B4C63 from the mixed micellar formulation co-encapsulating both drugs was sustained compared to free drugs [11]. However, the release of A83B4C63 was higher from the co-delivery systems compared to micelles encapsulating A83B4C63 alone, the release of SN-38 was lower from the co-delivery system compared to that of PM-SN-38 alone. [00164] We then investigated the synergy between SN-38 as free and polymeric micellar formulation with that of PM 26 /A83 formulation at different concentrations and ratios, first, using Combenefit ® software. Our data revealed a synergy between free SN- 38 and PM 16 -SN-38 with PM 26 /A83 at a concentration range of 0.01-1 µM for SN-38 and 10-40 µM for A83 (Fig 5). [00165] Thereafter, we assessed the efficacy for this combination formulation in human CRC cell lines while keeping the concentration of A83 fixed at 10 µM. The cytotoxicity data revealed significantly higher cell viability reduction for the combination treatments compared to their counterparts with SN-38 treatment only. Perhaps due to the efficient intracellular release of SN-38, all micellar SN-38 formulations showed significantly higher cell viability reduction than conventional irinotecan with or without PNKP inhibitor in human CRC cell lines. The effect of combination treatment with PNKP + TOP1 inhibitor was also observed in the increased DNA damage and mediators of apoptosis in CRC cells compared to their respective individual treatments with TOP1 inhibitor within 6 h. [00166] In both HCT116 and HT-29 CRC cell lines, the co-delivery of TOP1 and PNKP inhibitors using mixed micelles induced significant cytotoxicity, DNA damage and increase of apoptosis biomarkers compared to its counterpart with TOP1 inhibitor encapsulation only [4], while the PNKP enzyme involved in repairing the single strand DNA breaks by SN-38 similar DNA damaging agents. Ideally, PNKP catalyzes the restoration of 5'-phosphate and 3'-hydroxyl termini and allow DNA ligase to allow the rejoin the breaks. PNKP also ensures that 5'-OH termini are phosphorylated although its phosphatase activity was predominant over the kinase activity in strand breaks with both 3'-phosphate and 5'-OH termini. When TOP1-DNA “dead-end” complex is generated by SN-38, TOP1 enzyme was found to be attached to the DNA 3'-phosphate covalently through a tyrosine residue and subsequently released from the DNA by Tyrosyl-DNA Phosphodiesterase 1 or TDP1 for processing prior to ligation steps. Therefore, PNKP was found to participate in the correction 3'-phosphate and 5'-OH termini of the DNA breaks [22]. In case of regular single stranded DNA breaks, PNKP is not considered to be the underlying DNA repair enzyme unless TOP1 is inhibited by TOP1 inhibitor. Similarly in this study, the non-toxicity of both free and micellar PNKP inhibitors were evidenced by no cell viability reduction up to 40 µM dose after 72 h treatment and also further verified by no expression levels of cleaved PARP and γ-H2AX in CRC cell lines. Due to non-toxic behavior of our novel PNKP inhibitor, relative IC 50 concentration was not measurable up to 40 µM treating dose which was 3 times higher treating dose than the required dose (10 µM) for desired chemo-sensitization in this study. This observation was also in agreement with our previous in vivo study where neither systemic nor tissue toxicity was identified in healthy CD-1 mice receiving A83B4C63 up to 50 mg/kg dose intravenously either solubilized with the aid of Cremophor EL: Ethanol formulation or similar micellar formulation (PM 26 /A83) investigated in this study [20]. [00167] It appears that the co-delivery of A83B4C63 and SN-38 in one polymeric micellar formulation (PM 16 -SN-38:PM 26 /A83), potentiated the effect of A83B4C63. This was evident from similar cell viability at much lower ratios of A83 to that of SN-38 in co- delivery systems (PM 16 -SN-38:PM 26 /A83) in both cell lines compared to that of combination treatment (PM 16 -SN-38 + PM 26 /A83). [00168] 5. Conclusion [00169] Most importantly, the chemical conjugation of SN-38 to the hydrophobic core of self-associating PEO-poly(ester) micelles was successful with a higher w/w loading in this study, leading to a suitable water-soluble SN-38 formulation for systemic administration. Co-delivery of both TOP1 and PNKP inhibitors in PEO-poly(ester) based nano-carriers showed promising in vitro chemo-sensitizing synergy in human CRC cell lines. The overall findings demonstrate PM16-SN-38:PM26 as a co-delivery platform for a novel combination treatment option against CRC. [00170] Abbreviations [00171] BCL, α-benzyl carboxylate-ε-caprolactone; BSA, bovine serum albumin; CDCl 3 , deuterated chloroform; CMC, critical micellar concentration; CRC, colorectal cancer; DIC, N, N′-diisopropylcarbodiimide; DLS, dynamic light scattering; DMAP, 4- dimethylaminopyridine; DMEM/F12, dulbecco’s modified eagle medium and F12; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deoxy ribonucleic acid; DP, degree of polymerization; EPR, enhanced permeability and retention effect; Kcps, kilo counts per second; mPEO, methoxy-polyethylene oxide; mPEO-b-PBCL, methoxy-poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone); mPEO-b-PBCL/SN-38, SN-38- incorporated mPEO-b-PBCL micelle; mPEO-b-PCCL, methoxy-poly(ethylene oxide)-b- poly(α-carboxyl-ε-caprolactone); mPEO-b-PCCL/SN-38, SN-38-incorporated mPEO-b- PCCL micelle; MW, molecular weight; PDI, polydispersity index; RBC, red blood cell; SDS, sodium dodecyl sulfate; SN-38, 7-ethyl-10-hydroxy-camptothecin; TEM, transmission electron microscopy; THF, tetrahydrofuran; TOP1, topoisomerase I; ZP, zeta potential. 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[00196] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference. [00197] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
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