| Popis: |
Targeting DNA repair enzymes has attracted much attention in recent years to overcome the therapeutic resistance in cancer therapy. Inhibition of DNA repair enzymes can be used to make cancer cells sensitive to the DNA damaging effect of ionizing radiation or chemotherapy. In addition, the downregulation or mutation of specific DNA repair enzymes and/or tumor suppressor proteins in cancer cells can make them particularly more sensitive to the inhibition of DNA repair, a process known as “synthetic lethality”. Human polynucleotide kinase/phosphatase (PNKP) is a bifunctional DNA repair enzyme which phosphorylates DNA 5′-termini and dephosphorylates DNA 3′-termini that processes the ligation of damaged DNA termini. The inhibition of PNKP can make cancer cells more sensitive to DNA damage by ionizing radiation or Topoisomerase I inhibitors. Through siRNA library screening, a synthetic lethal partnership between loss of PNKP and tumor suppressor Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN). This inspired development of several small molecule inhibitors of PNKP. These newly synthesized PNKP inhibitors are not water-soluble, therefore not injectable. My research aim was to develop delivery systems at nanometer size range that can target a small molecule inhibitor of PNKP, known as A83B4C63 to the tumor while reducing their access to normal tissues. The nanocarriers were fabricated from self-associating block copolymers based on poly(ethylene oxide) (PEO) and poly(caprolactone) (PEO-PCL) or a-benzyl carboxylate substituted poly(caprolactone), abbreviated as PEO-PBCL. The developed nanocarriers were used for the encapsulation of A83B4C63 alone or with the active metabolite of irinotecan, i.e., SN-38. The developed formulations were characterized for their average diameter, polydispersity, morphology, loading properties, release profiles as well as sensitization of cancer cells to SN38 and/or ionizing radiation both in vitro and in vivo. To identify the binding affinity between intracellular PNKP and A83B4C63, a novel biophysical assay known as Cellular Thermal Shift Assay (CETSA) was developed and used. Maximum tolerated dose of A83B4C63 formulated with the aid of Cremophor EL:Ethanol (CE) and nanocarrier formulations was investigated in healthy CD-1 mice. The performed biochemical toxicity and immune histochemical experiments demonstrated that the intravenous (IV) administration of A83B4C63 in nanocarriers or CE form was not toxic up to the maximum examined dose of 50 mg/kg dose, although the nanocarrier injection was tolerated better by mice. The in vivo anticancer activity of the above formulations was also determined in colorectal cancer xenografts in mice either in PTEN negative model as monotherapy or in wild type model in combination with radiation therapy using the Small Animal Radiation Research Platform (SARRP). The results provided evidence for the anticancer activity of nanocarrier formulation of A83B4C63 as monotherapy in PTEN deficient HCT116 xenografts in mice. Inhibition of tumor growth was also observed as a result of combination of A83B4C63 nanocarriers with radiation therapy in wild type PTEN+ HCT116 xenografts in mice. This contrasted with the CE formulation of the PNKP inhibitor that did not show any activity, in vivo. The superior activity of the nano-formulation of A83B4C63 over CE formulation was attributed to the enhanced distribution of the drug to tumor site by its nanocarrier. A synergistic effect was also observed when nanocarriers of A83B4C63 were combined with SN-38 or its nano-formulation in CRC models, in vitro. The outcomes of this thesis have demonstrated the great feasibility of nano-delivery of a novel inhibitor of DNA repair for CRC therapy either as a single drug in PTEN deficient form or in combination with DNA damaging therapeutics. |
