Double strand DNA damage repair is increased in multiple myeloma (MM) and may be responsible for clonal evolution and resistance to chemotherapy. The enzyme poly [ADP-ribose] polymerase-1 (PARP-1) is integral to the DNA damage response and is involved in single strand repair as well as homologous recombination. DNA-dependent protein kinase (DNA-PK) is central to non-homologous end joining. Inhibition of PARP or DNA-PK have both been shown to lead to cytotoxicity in myeloma cells. In addition, we have shown that combined inhibition leads to synergistic cytotoxicity as well as enhancing the effect of alkylator chemotherapy. In order to efficiently deliver combined PARP/DNA-PK inhibition on the individual cell level, we developed a unique combined nanoparticle comprising olaparib (PARP inhibitor) and a prodrug of PI-103 (DNA-PK inhibition).
We first synthesized an SN2 lipase-labile phosphatidylcholine prodrug of PI-103. The chemical structure of the prodrug was confirmed by 1H-nuclear magnetic resonance, low resolution mass spectrometry and high resolution mass spectrometry. The prodrug is able to self-assemble into a phospholipid bilayer, which formed the outer layer of the nanoparticle. Olaparib becomes incorporated into the core of the colloidal suspension due to its hydrophobicity. For the particle to be active against MM cells it would fuse with the cell membrane and then be internalized. Subsequently the outer prodrug layer would be enzymatically cleaved and activated and olaparib would be released from the inner core. The hydrodynamic diameter of developed nanoparticles was found to be <150nm with -20mV electrophoretic potential values indicating their colloidal stability. These particles were able to incorporate very high concentration of drug molecules (> 85%) and exhibited an enzymatic release of ~30% for PI-103 and ~50% for olaparib at 24 hours under low pH conditions of 4.0.
We subsequently tested the ability of the combination nanoparticle to induce cytotoxicity in RPMI-8226 myeloma cells by treating cells with increasing doses in liquid culture. In a standard proliferative assay (3[H] thymidine uptake), the combination nanoparticle efficiently inhibited proliferation with an IC50 of 1.3microM. As a comparison, we analyzed the cytotoxicity of particles containing olaparib alone (nano-olaparib), PI-103 alone (nano-PI-103) or control nanoparticles without either drug. Control nanoparticles did not exert significant cytotoxicity. At a physiologic dose of 5microM, combination particles resulted in 96.7% inhibition in proliferation in comparison to nano-olaparib (55.3%, p=0.03) and nano-PI-103 (71.5%, p=0.03). In addition, at 5microM concentrations, combination particles resulted in significantly greater inhibition of proliferation than when cells were treated with both nano-olaparib particles and nano-PI-103 particles (84.9%, p=0.03). Treatment with combination nanoparticles resulted in a dose dependent increase in G2M arrest and rapid induction of apoptosis within 3 hours with a 5microM dose leading to a 15% absolute increase in Annexin+ cells. Immunofluorescent staining for γH2AX after 24-hour treatment with combination particles also showed a marked dose dependent increase, confirming that cytotoxicity was a result of double strand DNA damage. In order to establish that combined PARP and DNA-PK inhibition was safe in normal hematopoietic cells, we treated normal CD34+ cells with increasing doses of combination particles up to 5microM in a standard colony assay in methylcellulose medium supplemented with cytokines. Colony numbers were unaffected by particle treatment. Finally we safely treated an RPMI-8226 NOD/SCID/gamma null xenograft model of myeloma with 1.5g/kg of combination nanoparticles. Final results including a comparison with non-nanoparticle standard drugs will be presented at the meeting.
We present the proof of principle that our novel nanoparticle combined PARP/ DNA-PK inhibition is feasible and potently inhibits myeloma cell growth in-vitro but does not affect normal CD34+ cells. In the future, it is possible that this drug delivery platform could be utilized to efficiently deliver other synergistic drug combinations.
Patel: Celgene: Consultancy, Honoraria.
Asterisk with author names denotes non-ASH members.