Antisense Strategies Against Aberrant Signal Transduction Components in Cancer Cells
Protein Kinase A as a Target
In mammalian cells, cAMP acts through cAMP-dependant protein kinases (PKA). PKA is composed of two genetically distinct catalytic and regulatory subunits. The activating ligand, cAMP, binds the R subunit which results in a conformational change and dissociates the holoenzyme into an R2-(c-AMP)4 dimer and two free C subunits that are catalytically active. There are two types of PKA - type I (PKA-I) and type II (PKA-II) which share a common C subunit but contain distinct R subunits (RI and RII), respectively. There are four distinct R subunit isoforms - RIa, RIb, RIIa and RIIb and two C subunits isoforms - Ca and Cb. PKA-I is directly involved in cell proliferation, neoplastic transformation is required for G1>S transition of the cell cycle, and mediates the mitogenic signals of growth factors including EGF and TGFa. It is selectively overexpressed in the majority of cancers and correlates with poor prognosis in ovarian and breast cancer. Conversely, PKA-II is preferentially expressed in normal tissues is also involved in cell growth and differentiation. Embryonic stage, cells mainly contain RIa/PKA-I; mature cells mainly contain RIIa/PKA-II.
Antisense targeting, which is associated with cessation of growth and functions associated with differentiating RIa mRNA, effectively downregulates RIa and upregulates RIIb resulting in differentiation of leukemia cells and arrest of growth in cancer cells of epithelial origin. Downregulation of PKA-I by the site selective cAMP analog 8-Cl-cAMP leads to tumor suppression in vitro and in vivo, and is accompanied by decreased expression of oncogenes and growth factors. GEMâ231 (HYB0165) is a 18-mer mixed-backbone RNA/DNA hybrid phosphorothiroate oligonucleotide targeted to the human RIa subunit of PKAI, specifically binding to the NH2 terminus 8-13 codons. There is strong evidence that RNase H (an endonuclease which recognizes RNA: DNA duplex and cleaves the RNA at the duplex site), cleaves the specific mRNA at the duplex site inhibiting translation of mRNA and synthesis of protein.
In one prior human trial with GEMâ231 the maximum tolerated dose was 240 mg/m2 (MTD) as a 2-hour infusion twice a week (Mon, Th or Tue, Fri). With no side effects or adverse clinical events, plasma pharmacokinetics of GEMâ231 were linear with dose over an 18-fold dose range (20-360 mg/m2), and the plasma half-life was similar to that of other oligonucleotides at 1.34 + 0.34 hours (mean, SD). Comparisons of the results of the plasma concentrations, whether expressed as Cmax or AUC, suggest that dosing by body surface area (mg/m2) resulted in a more predictable relationship with dose than if the same patients were to have had doses calculated on a body weight (mg/kg) basis.
The distribution and elimination of a single-dose study of 35S-GEMâ231 was assessed in rats. The total body half-life of GEMâ231 (and metabolites) was approximately 2 weeks. Due to this slow clearance, it is anticipated that accumulation, and possibly cumulative toxicity, should occur when GEMâ231 is administered more frequently than monthly. However, in order to deliver a significant dose over a short period of time, and in turn achieve drug levels that may confer a cytostatic or cytotoxic effect, twice-weekly dosing has been chosen as one of the schedules for clinical evaluation.
Single doses of 360 mg/m2 have been established as safe in a dose-escalation phase I trial (Protocol 231-001) in which GEMâ231 was administered as a 2-hour intravenous infusion twice weekly. This study enrolled patients with incurable solid tumors, a population of patients similar to that to be studied in the present trial. Additional studies have determined the safety of escalating doses of GEMâ231 by infusions of 24-h duration, once or twice weekly. Studies on-going at Montefiore Medical Center are exploring continuous infusion regimens as well as combination regimens with taxanes – e.g., Phase I Clinical and Pharmacokinetic Study of GEMâ231 Given as a Continuous Infusion in Patients with Solid Tumors and Hematologic.
Multi-drug resistance (MDR) in cancer poses a major obstacle to the success of taxol chemotherapy. The mechanisms underlying taxol resistance are complex and may involve regulation of other cellular pathways regulating MDR-associated protein expression (e.g., p-glycoprotein). It has been shown that cyclic AMP (cAMP)-resistant mutants of the Chinese hamster ovary (CHO) and the mouse adrenal cortical carcinoma cells harboring defective regulatory (RI-a) subunits of the cAMP-dependant protein kinase (PKA) are more sensitive than wild-type cells to chemotherapeutic agents that are substrates for P-glycoprotein. In addition, a transfectant overexpressing mutant RIa cDNA shows similar increased sensitivity to these drugs. The altered drug sensitivity in the RIa mutants results from reduced expression of the MDR gene, suggesting that PKA may regulate its expression. The C (catalytic) subunit mutants have no effect on MDR or non-MDR drugs; however, the C subunit mutants, like the RIa mutants, exhibit decreased kinase activity and unresponsiveness to growth inhibition by cAMP. These results suggest that the increased multi-drug sensitivity in the PKA mutants results from alteration of the RI-a subunit and not the kinase activity, thus implying novel functions for the RI subunit.
Furthermore, centrally modified mixed-backbone oligonucleotides targeted at PKA RIa demonstrate synergistic growth-inhibiting activity with taxanes, as well as cooperative antitumor effects with taxol in nude mice. These preclinical data suggest that taxol may be combined with GEMâ 231, which is a mixed backbone RNA/DNA hybrid oligonucleotide targeted at the human RIa subunit of protein kinase A (PKA RI a), specifically binding to the NH2 terminus 8-13 codons. The expected cytotoxicity would be synergistic and down-regulation of RIa may provide for one way to modulate MDR-dependent drug cytotoxicity. Several in vitro and in vivo studies have also shown that antisense oligonucleotides targeting the RIa subunit of PKA alone cause cell growth arrest and differentiation. Phase I open-label safety study of escalating doses of taxol in combination with escalating doses of GEM 231 in Patients with Refractory Solid Tumors.
