Insert Therapeutics, Inc.

Purpose: Camptothecin (CPT) has potent broad-spectrum antitumor activity by inhibiting type I DNA topoisomerase (DNA topo I). It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with relapsed/refractory lymphoma, but with only modest activity. Recently, we have developed a cyclodextrin-based polymer conjugate of 20-(S)-CPT (IT-101). In this study, we evaluated the preclinical antilymphoma efficacy of IT-101 as compared with CPT-11.Experimental Design: We determined an in vitro cytotoxicity of IT-101, CPT-11, and their metabolites against multiple human lymphoma cell lines. In human lymphoma xenografts, the pharmacokinetics, inhibitions of tumor DNA topo I catalytic activity, and antilymphoma activities of these compounds were evaluated.Results: IT-101 and CPT had very high in vitro cytotoxicity against all lymphoma cell lines tested. As compared with CPT-11 and SN-38, IT-101 and CPT had longer release kinetics and significantly inhibit higher tumor DNA topo I catalytic activities. Furthermore, IT-101 showed significantly prolonged the survival of animals bearing s.c. and disseminated human xenografts when compared with CPT-11 at its maximum tolerated dose in mice.Conclusions: The promising present results provide the basis for a phase I clinical trial in patients with relapsed/refractory lymphoma.

Purpose: Tubulysins are naturally occurring tetrapeptides with potent antiproliferative activity against multiple cancer cell lines. However, they are also highly toxic in animal models. In order to improve the therapeutic index of this class of compounds, a nanoparticle prodrug of tubulysin A (TubA) was synthesized and evaluated in vitro and in vivo.Experimental Design: A thiol derivative of TubA was covalently attached to a linear, β-cyclodextrin based polymer through a disulfide linker (CDP-TubA). The polymer conjugate assembled into stable nanoparticles. Inhibition of tubulin polymerization and antiproliferative activity of the polymer conjugate were evaluated in vitro. The preclinical efficacy of CDP-TubA administered i.v. was evaluated in nude mice bearing s.c. implanted human HT29 colorectal and H460 non–small cell lung carcinoma tumors.Results: The IC50 of CDP-TubA (in Tub A equivalents) was 24, 5, and 10 nmol/L versus 3, 1, and 2 nmol/L for Tub A in NCI-H1299 (lung), HT-29 (colon), and A2780 (ovarian) cell lines, respectively. Tub A and the active thiol derivative were potent inhibitors of tubulin polymerization, whereas CDP-TubA showed minimal inhibition, indicating that target inhibition requires release of the peptide drug from the nanoparticles. The maximum tolerated dose of CDP-TubA was 6 mg/kg (in TubA equivalents) versus 0.05 mg/kg for TubA in nude mice. In vivo, a single treatment cycle of three weekly doses of CDP-TubA showed a potent antitumor effect and significantly prolonged survival compared with TubA alone.Conclusions: Cyclodextrin polymerized nanoparticles are an enabling technology for the safe and effective delivery of tubulysins for the treatment of cancer.

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.