Anthony J. Kozielski

9-nitro-camptothecin delays growth of U-937 leukemia tumors in nude mice and is cytotoxic or cytostatic for human myelomonocytic leukemia lines in vitro

Abstract:  The camptothecin derivatives 9‐nitro‐camptothecin (9NC) and 9‐amino‐camptothecin (9AC) inhibit similarly growth of HL‐60, KG‐1, and U‐937 cells in vitro, whereas growth of THP‐1 cells is inhibited by 9AC, but not by 9NC. Growth inhibition is accompanied by enlargement of cells which contain one (HL‐60, THP‐1) or more (KG‐1, U‐937) nuclei. Flow cytometry studies showed that 9NC‐treated HL‐60 and U‐937 cells accumulate in the S and G2 phases of the cell cycle; then they die by apoptosis, with the HL‐60 cells being more sensitive than U‐937 cells to 9NC. In contrast, 9NC‐treated KG‐1 and THP‐1 cells accumulate in S and G2 phases, but resist death by apoptosis. Of the cell lines tested, only U‐937 cells xenografted in nude mice generated subcutaneous myeloid tumors, which exhibited a delayed growth in the presence of 9NC. Further, 9NC‐treated advanced U‐937 tumors regressed temporarily, indicating that U‐937 cells consist of 9NC‐sensitive and 9NC‐resistant populations.

Structure-activity relationship of alkyl camptothecin esters.

Abstract: The cytotoxicity of camptothecin (CPT) esters 1–6 was measured. Like parental camptothecin, esters 2 and 3, but not 1, 4, 5, and 6, inhibited proliferation of human leukemia cells in culture and induced programmed cell death as assessed by flow cytometry studies. Exhibition of similar levels of antiproliferative activities of CPT 2 and 3 required different incubation time periods in cell cultures, with CPT and 3 requiring the shortest and longest periods, respectively. Both 2 and 3 were inactive against cells resistant to the semisynthetic CPT derivative 9‐nitrocamptothecin and unable to stabilize DNA‐topoisomerase I (Topo I) “cleavable complexes” in a cell‐free system, suggesting that Topo I activity was required but insufficient for the mechanism of action of 2 and 3. Mouse liver homogenate converted esters to parental CPT, but the conversion rates were different with different esters. Of four tested esters in this experiment, ester 2 had the fastest conversion rate. In vivo studies showed that ester 2 had an exceptional lack of toxicity in nude mice, even at enormous doses, and demonstrated extensive activity against human breast and colon tumors grown as xenografts in immunodeficient nude mice, whereas no antitumor activity was observed for the other esters. In conclusion, ester 2 is a prodrug of the antitumor compound CPT, and it can be administered at very high doses in mice with no appearance of toxicity. This study provides a basis for further evaluation of CPT ester 2 as an investigational anticancer agent.

Sensitivity of camptothecin-resistant human leukemia cells and tumors to anticancer drugs with diverse mechanisms of action.

Human leukemia U-937 cell clones resistant to 9-nitrocamptothecin (9NC) appear after exposure to increase 9NC-concentrations. Drug resistance is irreversible, regardless of whether the 9NC-resistant (U-937/CR150) cells grow in media with or without 9NC. U-937/CR150 cells are more sensitive than wild type U-937 (U-937/wt) cells to topoisomerase II-directed drugs, amsacrine, daunorubicin, and etoposide. The mitotic inhibitor, vincristine, induces hyperdiploidy in U-937/wt, but not in U-937/CR150 cells, whereas the antimetabolites, cytarabine and methotrexate, and the nitrosourea, carmustine, elicit similar responses in both U-937/wt and U-937/CR150 cells. U-937/CR150-generated tumors in nude mice are sensitive to etoposide. The clinical implications of increased sensitivity of 9NC-resistant tumors to some anticancer drugs are discussed.

Crystalline Camptothecin-20(S)-O-Propionate Hydrate:A Novel Anticancer Agent with Strong Activity against 19 Human Tumor Xenografts

To find a more effective and less toxic chemotherapeutic agent, we have successfully prepared crystalline camptothecin-20(S)-O-propionate hydrate (CZ48) by reacting camptothecin with propionic anhydride using concentrated sulfuric acid as catalyst. The biological effectiveness of this new anticancer agent was evaluated by using xenografts of human cancers in nude mice as the testing models. The extensive treatment of 21 human tumors with various dose levels of CZ48 has shown that this agent is highly effective against many different human tumors tested with a striking lack of toxicity. Of the 21 human tumor lines tested, 9 regressed, 5 were <10% of the control, 3 were <20%, and 2 were <40%. Two tumors did not respond. The total response rate was 90% (19 of 21). No toxicity was observed in mice. The effective doses required to achieve the positive response varied from 100 to 1,000 mg/kg/d depending on the tumors. The maximum tolerated dose was not reached because of the nontoxic nature of the drug in mice. Thus, this compound has a much wider therapeutic index compared with that of the existing anticancer drugs currently in use.

Influence of route of administration on [3H]-camptothecin distribution and tumor uptake in CASE-bearing nude mice: whole-body autoradiographic studies

Abstract Camptothecin (CPT) inhibits the growth of a wide variety of experimental tumors. As a part of our exploration of this drug for use as a cancer chemotherapeutic agent, we studied the effect of route of administration on the absorption, distribution and tumor uptake of [3H]-CPT. The rate of disappearance of [3H]-CPT-derived radioactivity from blood during the first 48 h was highest following oral than following intravenous (i.v.) administration. Thereafter blood levels were low irrespective of route of administration. Considerable [3H]-CPT-derived radioactivity was detected in urine and feces up to 48 h after dosing. Distribution studies were conducted using quantitative whole-body autoradiography (WBA). These studies revealed that independent of the route of administration, [3H]-CPT was rapidly excreted in the bile (gallbladder) followed by elimination into the small and large intestinal tract. Levels of CPT-derived radioactivity in the kidneys were minimal and mostly localized in the renal pelvis. Hepatic concentrations of CPT were low and were almost equal to those of the tumor. The lungs of animals treated i.v. showed higher uptake of radioactivity than those treated intramuscularly or orally. Tumor/blood ratios were slightly higher following oral administration than following administration by other routes. This study indicates that CPT is primarily eliminated via the bile. The gastrointestinal tract is the major site of accumulation and excretion of CPT.

Anticancer Activity of New Haloalkyl Camptothecin Esters against Human Cancer Cell Lines and Human Tumor Xenografts Grown in Nude Mice

All chemotherapeutic agents currently in use have a narrow window of therapeutic index of 1 to 1.2. Camptothecin ester compounds are reported to have a wider therapeutic index when being used to treat human xenografts in nude mice. As a continuous effort in searching for better chemotherapeutic agents for treating cancers, new haloalkyl camptothecin and 9-nitrocamptothecin ester derivatives 2a-b and 3a-d were prepared by respective acylation of camptothecin 1a and 9-nitrocamptothecin 1b with the corresponding acylating agents. These new derivatives were tested in vitro against 8 human cancer cell lines using 7 different concentrations ranging from 5 to 300 nM and also in vivo against various types of human tumor xenografts grown in nude mice. Most of these new compounds started showing inhibitory effects on the growth of 8 cancer cell lines at concentration of 80 nM and achieved greater than 70% inhibitions against these cell lines when the concentration increased to 300 nM. Compound 2a and 3a showed good activity against human tumor xenografts in nude mice. Compared to mother compound camptothecin, 3a was much less toxic in mice with a better therapeutic index, having the potential to be further developed as a safer treatment for cancers.

Abstract 2769: DSRCT: Establishing xenograft and tissue culture cell lines and assessing chemosensitivity.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, a younger and overall healthier patient population, and because it lacks definitive histologic and immunohistologic features, it is possible that DSRCT is frequently delayed in diagnosis or is entirely misdiagnosed as a different kind of abdominal sarcoma. This study seeks to rectify the dearth of models that exist for this rare neoplasm through the development of several DSRCT tissue culture and xenograft lines. Samples were received from surgeries and biopsies from patients around the world and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected onto the dorsal flank of nude mice. Of the 15 samples received, 9 were established into xenograft lines and 6 of those into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and two different growth rates among established xenograft lines. Chemosensitivity of tumors was assessed against a variety of chemotherapeutic agents and IC 50 values established for each drug and cell line. Interestingly, the cell lines were split into two distinct groups in terms of morphology, growth rate and chemosensitivity. Overall, the establishment of these xenograft and tissue culture lines gives researchers tools to evaluate DSRCT response to chemotherapy and investigate DSRCT-specific signaling pathways or mechanisms. Citation Format: Constantine SA Markides, Dana M. Vardeman, Linh Luong, Douglas R. Coil, Anthony Kozielski, John Mendoza, Beppino C. Giovanella. DSRCT: Establishing xenograft and tissue culture cell lines and assessing chemosensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2769. doi:10.1158/1538-7445.AM2013-2769