Preclinical results of camptothecin-polymer conjugate (IT-101) in multiple human lymphoma xenograft models

Abstract

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.

Figures

Fig. 1

(A) Plasma concentration of polymer-bound (■), free CPT (▲), CPT-11 (▼), and SN-38 (◆) as a function of time after a single bolus i.v. injection of IT-101 (10 mg/kg) and a single i.p. injection of CP-11 (100 mg/kg). (B) Tumor concentration of polymer-bound (■), free CPT (▲), CPT-11 (▼), and SN-38 (◆) as a function of time after a single bolus i.v. injection of IT-101 (10 mg/kg) and a single i.p. injection of CPT-11 (100 mg/kg). Points and error bars indicate mean and standard error of mean, respectively.

Fig. 2

Administration of IT-101 (10 mg/kg, i.v., single dose) can significantly inhibit tumor DNA Topo I catalytic activity in lymphoma xenografts as compared to CPT-11. DNA relaxation assay was done with nuclear extracts containing 0.5 μg total protein, isolated from s.c. lymphoma xenografts at four time points (before dosing, 2 hours, 24 hours, and 48 hours after dosing), as depicted. The mean percentage of tumor DNA Topo I catalytic activity inhibited by either IT-101 or CPT-11 was calculated from three or more separate experiments. Error bars indicate standard error of mean.

Fig. 3

Administration of IT-101 (5 mg/kg, i.v., qwk × 3) and (10 mg/kg, i.v., qwk × 3) to animals bearing three distinct s.c. human lymphoma xenografts can result in significant better survival as compared to CPT-11. Treatments were initiated at 10, 4, and 16 days after subcutaneous injection of Daudi cells, Karpas 299 cells, and L540 cells, respectively. Treatment arms included untreated control (■), CPT-11 (100 mg/kg, i.p., qwk × 3) (◇), IT-101 (5 mg/kg, i.v., qwk × 3) (○), and IT-101 (10 mg/kg, i.v., qwk × 3) (x). The tumor burden was monitored longitudinally by physical measurements. Using Kaplan-Meier method, the survival curves were plot for each treatment group and the log-rank test was used to compare the percent animal survival between treatment groups. In Daudi tumors and L540 tumors, a significant difference between the groups treated with either IT-101 (5 mg/kg, i.v., qwk × 3) or IT-101 (10 mg/kg, i.v., × 3) was not observed. However, these two treatment groups achieved significantly better survival as compared to the groups treated with CPT-11 (P < 0.0001 and P < 0.0001, respectively). In Karpas 299 tumors, no significant difference between the groups treated with either IT-101 (5 mg/kg, i.v., qwk × 3) or IT-101 (10 mg/kg, i.v., qwk × 3) was observed; but animals treated with IT-101 at (10 mg/kg, i.v., qwk × 3) had significantly better survival as compared to those treated with CPT-11. (P = 0.0072). Points indicate mean survival.

Fig. 4

Administration of IT-101 (5 mg/kg, i.v., qwk × 3) and IT-101 (10 mg/kg, i.v., qwk × 3) in disseminated human B-cell lymphoma xenograft-bearing NOD/scid mice could result in significant better survival as compared to CPT-11. Treatments were initiated on 11 days after tumor inoculation. The treatment arms included untreated control (■), CPT-11 (100 mg/kg, i.p., qwk × 3) (◇), IT-101 (5 mg/kg, i.v., qwk × 3) (○), and IT-101 (10 mg/kg, i.v., qwk × 3) (x). The tumor burden was monitored longitudinally by quantification of tumor-derived ffLuc-activity. (A) Total photon flux normalized for exposure time and surface area and expressed in units of photons/second/cm2/steradian (p/s/cm2/sr) for individual mice was graphed over time and serial pseudocolor images representing light intensity from Daudi+ ffLuc in selected mice were shown. (B) Kaplan-Meier survival curves were plot for each treatment group. The log-rank test was used to compare the percent animal survival between treatment groups. A significant difference between the groups treated with either IT-101 (5 mg/kg, i.v., qwk × 3) or IT-101 (10 mg/kg, i.v., qwk × 3) was not observed; but these two treatment groups could significantly establish better survival as compared to the animals treated with CPT-11 (P = 0.0002 and P < 0.0001, respectively). Points indicate mean survival.

Fig. 5

IT-101 (10 mg/kg, i.v., qwk × 3) had superior antilymphoma activity as compared to IT-101 (5 mg/kg, i.v., qwk × 3) and CPT-11 in disseminated human anaplastic large T-cell xenografts. NOD.scid/NCr mice were intravenously injected with ffLuc+ Karpas 299 cells to establish disseminated human anaplastic large T-cell xenografts and treated with the indicated cytotoxic agent beginning eight days later. Treatment arm included untreated control (■), CPT-11 (100 mg/kg, i.p., qwk × 3) (◇), IT-101 (5 mg/kg, i.v., qwk × 3) (○), and IT-101 (10 mg/kg, i.v., qwk × 3) (x). The tumor burden was monitored longitudinally by quantification of tumor-derived ffLuc-activity. (A) Total photon flux normalized for exposure time and surface area and expressed in units of photons/second/cm2/steradian (p/s/cm2/sr) for individual mice was graphed over time and serial pseudocolor images representing light intensity from Karpas 299+ ffLuc in selected mice were shown. (B) Kaplan-Meier survival curves for each treatment group. The log-rank test was used to compare the percent animal survival between treatment groups. IT-101 (10 mg/kg, i.v., qwk × 3) treated animals had significantly longer survival as compared to those treated with IT-101 (5 mg/kg, i.v., qwk × 3) and those treated with CPT-11 (P = 0.0009, P = 0.0049, respectively). Points indicate mean survival.