Structure-activity relationship of alkyl camptothecin esters.

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.

Alkyl esters of camptothecin and 9-nitrocamptothecin: synthesis, in vitro pharmacokinetics, toxicity, and antitumor activity.

Eleven camptothecin esters, 6a-e and 7a-f, were prepared by straightforward acylation of camptothecins with the corresponding acylating reagents such as organic anhydrides and carboxylic acid chlorides. The in vitro pharmacokinetic determination of lactone levels of esters 6a and 7b showed that the biological life span of their lactone forms in human and mouse plasma significantly increased when compared with their mother compounds, camptothecin (3) and 9-nitrocamptothecin (4). The differences of lactone levels between human plasma and mouse plasma for 6a and 7b were much smaller than what was observed for their mother compounds. The in vivo antitumor activity and toxicity studies demonstrated that some of these esters were very active against human tumor xenografts in nude mice and had an exceptional lack of toxicity in nude mice, even at enormous doses.

Comparative disposition of the antineoplastic agent 9-nitrocampotothecin and the inactive isomer 12-nitro camptothecin in CASE-bearing nude mice: effect of route of administration on tissue distribution.

PURPOSE: 9-Nitrocamptothecin (9-NC) and 12-nitrocamptothecin (12-NC) are synthetic structural analogues of camptothecin (CPT) which have been prepared to explore the structure/activity relationship of this group of compounds against a wide variety of experimental tumors. As part of our investigation of the pharmacology and the mechanism of tumor inhibition of these compounds, we examined the effect of route of administration on the distribution of tritium-labeled 9-NC and 12-NC, an active and a poor chemotherapeutic agent, respectively. METHODS: Quantitative whole-body autoradiography was used and our results were compared with previous results obtained with the parent compound CPT. RESULTS: These studies revealed that, independent of the route of administration, both CPT derivatives were rapidly distributed to gall bladder, gastrointestinal tract and kidney. The excretion from these organs was indicated by the high levels of radioactivity in urine (urinary bladder) and feces (large intestines). The studies also indicated that the distributions of 9-NC and 12-NC were qualitatively similar, but quantitatively higher uptake of radioactivity was observed in animals treated with 12-NC than in those treated with 9-NC at 30 min following treatment. With the exception of the late sampling time (12 h after administration), the accumulation of radioactivity in the lungs (bronchioles) of animals that received an intravenous (i.v.) dose of 9-NC or 12-NC was higher than those treated with an intramuscular (i.m.) dose. However, the retention of drug-derived radioactivity in the tumors of mice treated with an i.m. dose of 9-NC was higher than that in the tumors of i.v.-treated animals and was also higher than that in tumors of animals treated with 12-NC. CONCLUSIONS: These results suggest that higher accumulation of 9-NC in tumor tissues than of 12-NC may contribute to the more potent chemotherapeutic activity of the former agent. Our results also suggest that i.m. injection is a more effective route of administration than i.v. administration.

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

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.

Growth inhibition of human cancer metastases by camptothecins in newly developed xenograft models.

Several metastatic models have been developed using clonal selection of human malignant cells metastasizing into a specific organ in NIH-I Swiss immunodeficient mice. The organs of choice were the central nervous system (CNS), targeted by metastases of malignant melanoma, and the liver, with metastases of colon adenocarcinoma. Additional models of adrenal metastases by malignant melanoma, and CNS involvement by implanted human lung squamous carcinoma or lymphoblastoid cells, are also available. Organ metastases, as well as the effects of treatment, were confirmed by autopsies and histological examination of the tissues or by a surgical inspection of the liver. The treatment end points were established as the increases in survival times of treated mice relative to placebo-treated controls. Camptothecins injected i.m. or delivered via gastrointestinal tract inhibit the growth of CNS metastases and increase the survival of treated animals. 9-Amino-20(S)-camptothecin was effective in the CNS model and in the model of liver metastases. The drug increased 3.3- and 5.7-fold the survival rates relative to untreated controls with metastases of colon adenocarcinoma to the liver, and all camptothecins were significantly more effective than 5-fluorouracil, currently a drug of choice in treatment of this disease. The xenograft models of metastases are available for studies of drug passage through the blood-brain barrier optimization of drug delivery to the liver, and for the development of new camptothecin-based treatment strategies.

Modulation of the response to chemotherapy in a human melanoma clone by the site of growth in the nude mouse.

Organ environment has been shown to modulate the efficacy of chemotherapy. A nude mouse model was used to study the influence of the in vivo environment on the sensitivity to chemotherapy of a human melanoma. A melanoma clone, SB1A, was implanted in two different sites (adrenals and subcutis) of nude mice which were subsequently treated with high-dose cyclophosphamide. After treatment, a 42% complete response rate was observed for the adrenal implants. No response was seen in the subcutis despite the same chemotherapy administration. This result is not attributable to differences in drug concentration in the target sites, because of the high-dose chemotherapy used. This model shows that the in vivo environment modifies the sensitivity to chemotherapy of a malignant melanoma clone. Further research is needed to clarify the role of host factors and of specific cell-stroma interactions that modify the biological behavior of malignant cells.

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.

Therapeutic efficacy of camptothecin derivatives against human malignant melanoma xenografts.

Camptothecin (CPT) and some of its derivatives are currently used in several clinical studies with patients bearing leukaemias, lymphomas, and malignancies of various solid tissues. Therefore, it is important to establish parameters and conditions that will allow the drugs to exhibit maximal anti-cancer effectiveness with minimal toxic effects. We tested several water-insoluble CPT derivatives for their ability to inhibit growth of human melanoma tumours xenografted in nude mice. We found that anti-tumour effectiveness and drug-induced toxicity depended on (a) the CPT derivative; (b) the drug dose administered; (c) the mode of administration; and (d) the scheduling of drug administration. For all practical purposes, oral administration of the CPT derivative, 9-nitrocamptothecin, has produced the best overall results.

Regression of human breast carcinoma tumors in immunodeficient mice treated with 9-nitrocamptothecin: differential response of nontumorigenic and tumorigenic human breast cells in vitro.

We have shown recently that the plant alkaloid camptothecin and its derivatives inhibited growth of human carcinoma and melanoma cells in vitro and induced regression of advanced human malignant melanoma tumors growing in immunodeficient (nude) mice. Here, we have extended these studies to show that the camptothecin derivative 9-nitro-20(S)- camptothecin (9NC) induces complete regression of advanced breast carcinoma tumors growing in nude mice. We also report that 9NC inhibits growth of nontumorigenic and tumorigenic breast cells in vitro. However, flow cytometry studies show that 9NC elicits differential effects on the cell cycle of nontumorigenic and tumorigenic cells. In general, 9NC-treated nontumorigenic cells accumulate slowly at the G2 phase of the cell cycle with no cell death. In contrast, 9NC-treated tumorigenic cells transverse rapidly from G1 to S phase followed by cell death. Removal of 9NC from the cell cultures resulted in most nontumorigenic cells dividing, whereas tumorigenic cells continued to die after removal of 9NC. Taken together, the findings indicate a different response of nontumorigenic and tumorigenic breast cells to 9NC.

Camptothecin derivatives induce regression of human ovarian carcinomas grown in nude mice and distinguish between non-tumorigenic and tumorigenic cells in vitro.

We have recently shown that the plant alkaloid 20(S)-camptothecin and its derivatives 9-nitro-20(S)-camptothecin(9NC) and 9-amino-20(S)-camptothecin(9AC) inhibit the growth of a variety of human tumors xenografted in nude mice. In this report, we demonstrate that 9NC and 9AC effectively inhibit growth, and subsequently induce regression, of human ovarian tumors grown in nude mice. Tumor regression is accompanied by degenerative changes in the tumor cells as assessed by microscopic observations of histological sections prepared from the tumors. Parallel experiments in vitro show that 9NC inhibits in a similar manner the growth of human ovarian carcinoma cells, regardless of their ability to induce tumors when xenografted in nude mice, and induces similar morphological changes in both non-tumorigenic and tumorigenic cells, as assessed by microscopic observation. Flow cytometry studies show that 9NC-induced growth inhibition of the non-tumorigenic cells is associated with accumulation of these cells in G2. In contrast, 9NC-induced growth inhibition of the tumorigenic cells is associated with the generation of cells containing a reduced DNA content, that is, cells programmed to die. In conclusion, camptothecins appear to be cytostatic for non-tumorigenic, but cytotoxic for tumorigenic cells, an important finding from viewpoints of cell biology, pharmacology and cancer chemotherapy.

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.

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.

Efficacy of camptothecin congeners in the treatment of human breast carcinoma xenografts.

We have shown recently that the plant alkaloid camptothecin and some of its derivatives inhibit growth of human breast carcinoma cells in vitro and induce complete regression of human breast tumors grown in nude mice. Because of the use of camptothecin derivatives in several clinical studies with patients bearing various types of cancer, in this report, we have investigated and described parameters and conditions that can modulate the anticancer effectiveness and cytotoxicity of these drugs when administered as suspensions. It is demonstrated that the antitumor effectiveness and drug-induced toxicity depend on the camptothecin derivative, the drug dose administered, the route of administration, and the scheduling of drug administration. 9-aminocamptothecin administered i.m. generates the best results, but for all practical purposes, 9-nitrocamptothecin administered orally appeared to be the camptothecin derivative and route of administration of choice. Further, we report the partial response of one breast tumor to camptothecin and propose that this tumor may require chemotherapy with a camptothecin derivative followed by an anticancer drug with a different mechanism of cell-killing activity.

Complete inhibition of growth followed by death of human malignant melanoma cells in vitro and regression of human melanoma xenografts in immunodeficient mice induced by camptothecins.

The plant alkaloid camptothecin and three camptothecin derivatives were used to study responses of human malignant melanoma (BRO) cells xenografted in immunodeficient (nude) mice. Camptothecin and its derivatives 9-nitro-20(S)-camptothecin and 9-amino-20(S)-camptothecin inhibited growth of tumors and caused regression in all tumor-bearing mice. Tumor regression was accompanied by degenerative changes in the tumor cells, as assessed by microscopic observations of histological sections prepared from the tumors. No toxic effects were observed in the drug-treated mice, with or without xenografts. In parallel experiments, camptothecin, 9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin inhibited proliferation of BRO cells in vitro and resulted in dramatic morphological cellular changes comparable to those observed in the sections of solid tumors. The derivative 12-nitro-20(S)-camptothecin had no effect on BRO tumors or cell cultures. The difference between 9-nitro-20(S)-camptothecin and 12-nitro-20(S)-camptothecin is the position at which the NO2 group is attached to the camptothecin molecule. In contrast to BRO melanoma cells, none of the camptothecin derivatives had any effect on cultured human melanocytes, the normal counterparts of melanoma cells. Taken together, the findings indicate that camptothecin and derivatives exert different effects on the growth and morphology of normal and malignant (BRO) melanocytes.

Complete growth inhibition of human cancer xenografts in nude mice by treatment with 20-(S)-camptothecin.

20-(S)-Camptothecin (CAM), a plant alkaloid, was tested against 13 human cancer xenograft lines carried by immunodeficient (nude) mice. The drug, formulated in 20% intralipid and given i.m., was more effective than any other clinically available drug tested. It was found that: (a) CAM, at nontoxic doses, suppressed growth and induced regression of cancer of the colon (3 lines), lung (4 lines), breast (2 lines), stomach (1 line), ovary (1 line), and malignant melanoma (2 lines); (b) the drug was equally effective administered i.m. or p.o. Both routes are significantly better than i.v. administration; (c) CAM is substantially more effective and less toxic than its sodium salt, which was unsuccessfully tested in cancer patients. CAM should be further tested against responsive cancers as a drug which is easy to isolate and formulate for large-scale studies.

Specific organ metastases of human melanoma cells injected into the arterial circulation of nude mice.

The purpose of this study was to examine whether organ specific metastasis can be produced by different subpopulations of cells isolated from a single human melanoma. Three clones, SB1, SB2, SB3, and a variant line SB1 Asc (obtained from ascites fluid after i.v. inoculation of SB1 cells in a nude mouse) - all isolated from a primary human cutaneous melanoma - were inoculated into the left ventricle (i.c.) or intravenously (i.v.) into nude mice. Three to 16 weeks later, the animals were killed and autopsied. The clones produced different patterns of metastasis. Three to 4 weeks after i.c. inoculation of 2 x 10(6) cells, cells of clone SB1 produced numerous rapidly progressing metastases in the brain, whereas cells of SB1 Asc produced large metastases in the adrenals and sometimes in the ovaries. Cells of clones SB2 and SB3 produced a few small metastases without any characteristics pattern. The initial arrest of tumor cells in various organs did not predict the outcome of metastasis. This conclusion is based on studies of distribution and fate of tumor cells labeled with 111Indium Oxine. Therefore, organ specific metastasis produced by human melanoma cells inoculated i.c. into nude mice is likely to depend on the interaction of unique metastatic cells with organ environment.