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.

9-Nitrocamptothecin liposome aerosol treatment of human cancer subcutaneous xenografts and pulmonary cancer metastases in mice.

The purpose of this study was to test the anticancer properties of the water-insoluble derivative of camptothecin, 9-nitrocamptothecin (9NC), administered in a liposome formulation (L-9NC) in aerosol to mice with subcutaneous xenografts of three human cancers and in mice with murine melanoma and human osteosarcoma pulmonary metastases. The drug was formulated with dilauroylphosphatidylcholine and nebulized in particle sizes of 1.2-1.6 microns mass median aerodynamic diameter and a geometric standard deviation of 2.0. The aerosol was generated with the nebulizer flowing at 10 l/min and delivered to mice in sealed plastic cages or in a nose-only exposure chamber. Aerosol was administered for 15 min to 2 hr daily, delivering deposited doses in the respiratory tract of 8.1-306.7 micrograms of 9NC/kg. With subcutaneous tumors, growth was greatly inhibited or tumors were undetectable after several weeks of treatment. We also showed that oral dosage with L-9NC had no detectable effect on cancer growth, and thus the benefit from aerosol treatment was due to pulmonary deposition and not the larger fraction of drug deposited in the nose of mice during aerosol treatment which is promptly swallowed. Intramuscular L-9NC in slightly larger doses than given in the aerosol had detectable anticancer activity, but it was significantly less than in mice receiving the drug by aerosol. With metastatic pulmonary cancers, treated animals showed highly significantly less cancer growth than control animals. L-9NC aerosol showed a major therapeutic benefit in the treatment of subcutaneous human cancer xenografts in nude mice, suggesting that cancers at systemic sites might be responsive to this treatment. In addition, the strong anticancer effect of L-9NC aerosol on pulmonary metastases offers a therapeutic approach for treatment of pulmonary cancers. Thus, L-9NC aerosol may have applicability in the treatment of cancers throughout the body.

Modified lactone/carboxylate salt equilibria in vivo by liposomal delivery of 9-nitro-camptothecin.

The lactone stability of camptothecins is critical for their anticancer activity. A stable liposomal 9-nitro-camptothecin formulation was developed to circumvent the drawbacks of low aqueous solubility and lactone instability and to provide sustained release of the agent in blood circulation. The potential merits of the formulation were demonstrated by its profoundly improved lactone stability in vivo, favorable pharmacokinetic and biodistribution characteristics in rats, and enhanced preclinical efficacy in tumor-bearing athymic mice.

Pharmacology of camptothecin esters.

An intact lactone ring of camptothecins is a structural requirement for their anticancer activity. Propionate esters of camptothecin (CPT) and 9-nitrocamptothecin (9NC), CZ48 and CZ112, respectively, have been synthesized as derivatives resistant to lactone hydrolysis and are chemotherapeutically active. In this study, we have examined the mechanism of action of CZ48 and CZ112 and their distribution, metabolism, and toxicity. CZ112 incubated in human plasma retained its lactone structure longer than 9NC (t1/2: 10.5 and < 1 hr for CZ112 and 9NC, respectively). This resistance to lactone hydrolysis was also observed in mouse plasma or albumin solutions. Neither CZ48 nor CZ112 inhibit topoisomerase I and thus are prodrugs dependent on hydrolysis to CPT or 9NC, respectively. Rates of hydrolysis of CZ48 to CPT are higher by homogenates of mouse liver, spleen, lung, and kidney than by plasma. Rates of hydrolysis by tumor cells in culture vary and were higher by breast cancer and melanoma cells than by colon cancer cells. On the basis of these and other data, it is proposed that CZ48 and CZ112 may act as anticancer agents by resisting hydrolysis to camptothecins while in circulation. Hydrolysis in tissues may release intact lactone in target tissues.

Alternative administration of camptothecin analogues.

The binding of camptothecin (CPT) to the DNA-topoisomerase complex is reversible, but it needs to be maintained for maximal inhibitory activity. It is also dependent on the chemical structure of CPT. The lactone form is thought to be necessary for the activity. In human serum, the equilibrium between lactone and carboxylate is in favor of the latter. For these reasons, alternative administration of CPT analogues is being evaluated. The ideal compound would remain in lactone form and would expose the host for long periods of time to its effects. Oral administration of irinotecan (CPT-11) and topotecan (TPT) is discussed by other investigators. We studied oral rubitecan and reported a low lactone to total drug area under the plasma concentration-time curve (AUCP) ratio (14.7%), with low plasma concentration over time despite repeated administrations and the presence of an enterohepatic cycle. Aerosolization of a liposomal formulation of rubitecan is currently under study. Six patients have been treated once a day for 5 days every 3 weeks. The dose was 6.7 micrograms/kg/day. Plasma levels are dose for dose higher than those after oral administration, but the ratio of lactone versus total drug is low. No toxicity was observed. The study will continue with increasing doses and lengths of administration. Intrathecal administration of topotecan has been studied in a phase I trial in children. Doses of 0.4 mg are tolerated without toxicity, and clinical responses have been seen in patients with refractory meningial carcinomatosis. Phase II studies are planned. Intraperitoneal (i.p.) administration of topotecan has been studied in a phase I trial as a 24-hour infusion in 5% dextrose at pH 3.5 every 21 days. Dose-limiting toxicity is 4 mg/m2. Toxic effects are neutropenia, anemia, emesis, fever, and pain. Five of 10 patients with ascites had symptomatic relief. Pharmacokinetic analysis demonstrates a second-order kinetics with elimination half-lives of 0.49 and 2.7 hours. The peritoneal to plasma AUC ratio was 31.2. Intramuscular, transdermal, and subcutaneous administrations have been extensively studied in the mouse.

Dependence of anticancer activity of camptothecins on maintaining their lactone function.

Camptothecins contain a lactone ring that exists in the closed form below ph 7. Above 7, the open (CPT+) and the closed (CPT) form coexist in a 50-50 ratio in mouse plasma and in a 90-10 ratio in human plasma due to the high affinity of human serum albumin (HSA) for CPT+. CPT+ is much less toxic than CPT and it is excreted much faster. In complete RPMI 1640 culture medium, the equilibrium CPT(+)-CPT is 50-50. If 4% HSA is added, it moves to 90-10 modeling for the human physiological situation.

Anticancer effect of 9-nitrocamptothecin liposome aerosol on human cancer xenografts in nude mice.

PURPOSE: To test the anticancer properties of the water-insoluble derivative of camptothecin, 9-nitrocamptothecin (9-NC) against human breast, colon and lung cancer xenografts in nude mice when administered in liposome aerosol. METHODS: The drug was formulated with dilauroylphosphatidylcholine and nebulized in a particle size of 1.6 microm +/- 2.0 mass median diameter to deliver doses of usually less than 200 microg/kg daily, 5 days per week. 9-NC liposome aerosols were generated with a Aerotech II nebulizer (CIS-USA) flowing at 101/min from a compressed air source and delivered to mice in sealed plastic cages or in a nose-only exposure chamber. RESULTS: Tumor growth was greatly reduced or tumors were undetectable after several weeks of treatment. Colon tumor was least responsive. 9-NC was better than the parent compound, camptothecin, also water-insoluble, tested by aerosol in a similar liposomal preparation. Equivalent doses of 9-NC liposome preparations administered by mouth were substantially without effect while there was some effect, but limited, of the liposome preparation given intramuscularly. CONCLUSIONS: 9-NC liposome aerosol was strikingly effective in the treatment of three human cancer xenografts growing subcutaneously over the thorax in nude mice at doses much smaller than those traditionally used in mice administered by other routes.

A phase II clinical and pharmacological study of oral 9-nitrocamptothecin in patients with refractory epithelial ovarian, tubal or peritoneal cancer.

9-Nitrocamptothecin (9-NC) is a water-insoluble topoisomerase I inhibitor with a broad antitumor activity in animal models. A phase II study was performed in patients with heavily refractory ovarian, tubal or peritoneal cancer (median number of previous chemotherapy regimens > 3) to determine the activity of a daily oral dose of 9-NC. 9-NC dose was 1.5 mg/m2/day for four consecutive days every week. Increments of 0.25 mg/day were authorized in patients without significant side effects. Of 29 evaluable patients, a 7% remission rate was observed. Thirty-four percent of patients had stable disease. The median survival was 8 months. Toxicity was evaluated in 31 patients. Grade 3 or 4 hematologic toxicity consisted of anemia in 10 patients (32%), neutropenia in eight (26%) and thrombocytopenia in three (10%). Grade > or = 2 non-hematologic toxic effects were nausea and vomiting in 26 (84%), diarrhea in 12 (39%), weight loss in seven (22%), chemical cystitis in six (19%) and neutropenic sepsis in six (19%). 9-NC was tolerated for sustained periods of time in some patients (up to 47 weeks). The observed 8-month survival in such a refractory patient population is noteworthy. Further clinical research of prolonged exposure to less toxic analogs of 9-NC is warranted.

A study of 9-nitrocamptothecin (RFS-2000) in patients with advanced pancreatic cancer.

This ongoing study evaluates the efficacy of oral 9-nitrocamptothecin (9NC), or RFS-2000, in the treatment of advanced pancreatic cancer. Patients received 9NC orally for 5 days/week; 8 weeks of therapy is required to achieve minimum effective dose. Starting dose was 1.5 mg/m2/day, with adjustments made as necessary. Patients were analyzed for changes in tumor size by CT scan, changes in serum CA 19-9 tumor marker levels, quality of life, and survival. 107 consecutive patients with advanced adenocarcinoma of the pancreas were enrolled before November 3, 1997. Of this group, 47 patients did not receive the minimum 2 courses of treatment necessary to induce response, leaving 60 evaluable patients. Primary dose-limiting toxicities were myelosuppression and interstitial cystitis. No deaths were attributed to 9NC. Median survival was 6.5 months for the 107 total patients and 8.7 months for the 60 evaluable patients, with one patient surviving at 44+ months. Of the 60 evaluable patients, 31.7% were responders (median survival 18.6 months; range 6.5-44.7+ months), 31.7% were stable (median survival 9.7 months), and 36.6% were non-responders (median survival 6.8 months). Fifty-seven previously untreated patients had a median survival of 7.3 months compared to 4.7 months for the 50 previously treated patients. Thirty-three patients who failed gemcitabine therapy prior to 9NC treatment had a median survival of 4.7 months. 9NC is safe and efficacious as first-line therapy for the treatment of advanced pancreatic cancer. It also shows some modest success as second-line therapy in treating gemcitabine failures.

A phase I clinical and pharmacological study of oral 9-nitrocamptothecin, a novel water-insoluble topoisomerase I inhibitor.

9-Nitrocamptothecin (9NC) is a water-insoluble topoisomerase I inhibitor with a broad antitumor activity in animal models. To determine the maximum tolerated oral dose (MTD), a phase I study was performed in patients with advanced cancer refractory to conventional chemotherapy. 9NC was administered orally with escalating doses to cohorts of five patients beginning at 1 mg/m2/day for five consecutive days every week for 4 weeks. Increments were 0.5 mg/m2/day for each cohort. Toxicity was evaluated in 28 patients diagnosed with various malignancies. Seven patients received 1 mg/m2/day for 28 weeks; 10 patients, 1.5 mg/m2/day for 68 weeks; and 26 patients, 2 mg/m2/day for 159 weeks. At 1.5 mg/m2/day or higher, the dose-limiting toxicity was hematologic, with grade 4 anemia in eight (29%); neutropenia in seven (25%) and thrombocytopenia in five (18%). Grade 2 or higher toxic effects occurred at each dose level: nausea and vomiting in 15 (54%), diarrhea in nine (32%), chemical cystitis in seven (25%), neutropenic sepsis in six (21%) and weight loss in five (18%) (N=28). Responses were observed after 2-8 weeks of therapy in five patients with pancreatic, breast, ovarian and hematologic tumors. Fourteen patients had a disease stabilization and one patient received treatment up to 18 months. The MTD of 9NC given orally has been estimated at 1.5 mg/m2/day for five consecutive days weekly. 9NC may be tolerated for sustained periods of time, but has the potential for significant hematologic, gastrointestinal and urinary bladder toxicity. Significant antitumor activity was observed, warranting further clinical investigations.

Altered sensitivities to anticancer and differentiation agents in etoposide-resistant human myeloid leukemia U-937 cells.

We previously have exposed U-937 human leukemia cells to stepwise increased concentrations of the anticancer drug etoposide, and this treatment has resulted in stable sublines (termed U-937/RE) exhibiting various extents of resistance to the drug and constitutively expressing c-fms mRNA, a specific marker of monocytic differentiation. In this report, we pursued studies to show that the P-glycoprotein blocker, verapamil, partially restores sensitivity to etoposide in U-937/RE cells. Further, the U-937/RE cells exhibit differential sensitivities to compounds that induce maturation of U-937 cells, as judged by the ability to reduce nitroblue tetrazolium and by morphologic changes, and increased sensitivities to apoptosis induction by the cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) and the anticancer drugs 9-nitrocamptothecin and doxorubicin. In addition, the U-937/RE cells, xenografted in immunodeficient mice, demonstrate decreased or no ability to induce tumors. Taken together, these findings indicate that U-937/RE cells differ from the parental U-937 cells in several functional properties and can serve as models to develop protocols for treatment of human leukemia.

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.

PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.

Mapping of homozygous deletions on human chromosome 10q23 has led to the isolation of a candidate tumor suppressor gene, PTEN, that appears to be mutated at considerable frequency in human cancers. In preliminary screens, mutations of PTEN were detected in 31% (13/42) of glioblastoma cell lines and xenografts, 100% (4/4) of prostate cancer cell lines, 6% (4/65) of breast cancer cell lines and xenografts, and 17% (3/18) of primary glioblastomas. The predicted PTEN product has a protein tyrosine phosphatase domain and extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions. These homologies suggest that PTEN may suppress tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through interactions at focal adhesions.

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.

Pharmacokinetics of camptothecins administered orally.

The equilibrium between lactone and salt forms of camptothecin (CPT) and its derivatives including 9-nitrocamptothecin (9-NC) depends on pH, binding to albumin and other factors. Their antitumor activity is associated with the lactone form. Our goal was the development of dosing regimens optimal for chemotherapeutic activity of the drug. The effect of p.o., i.v. and i.m. administration on the tumor uptake of [3H]-CPT or [3H]-9-NC in tumor-bearing nude mice was studied by whole-body autoradiography. In all cases, [3H]-CPT or [3H]-9-NC accumulated mainly in the gastrointestinal tract. Comparatively lower levels of drug were detected in liver, kidney, tumor, and other sites. Consistently high tumor/blood ratios following oral administration of drug suggest this route as the most effective way of treatment. Within 4 h of i.s. administration of 2 mg/kg CPT or 1 mg/kg 9-NC to mice, lactone forms were 57-81% and 47-95% of total plasma drug levels, respectively. However in plasma of humans treated p.o. with varying doses of CPT or 9-NC, lactone forms were only a minor component of total drug levels. It is concluded that ratios of lactone/total drug are much higher in mice than in humans, which influence the therapeutic efficacies these drugs in the two species.

Protocols for the treatment of human tumor xenografts with camptothecins.

Thirty-five human tumors of various histological types xenografted at various sites into nude mice and rats have been used to assess the anticancer activity of camptothecin and derivatives administered by different routes (subcutaneous, intramuscular, intravenous, intrastomach, and transdermal). Camptothecins are active against human tumors at every site including the brain. So far, the best anticancer/toxicity ratio has been found with 9-nitrocamptothecin (9NC) and 9-aminocamptothecin (9AC) to which 9NC converts in the body of mammals. Comparing the results obtained during clinical trials with the animal ones, it is evident that camptothecins are much less active in humans than in mice against human tumors. This is probably due to the fact that in humans the lactone ring of camptotecins opens much faster than in mice. Measurement of the area under the curve (AUC) in mice and humans under comparable conditions of administration gives values of 3% closed lactone for man versus 55% in mice for 9NC. Clearly this is the crucial problem to overcome in order to improve the efficacy of the camptothecins as anticancer agents.

Phase I clinical and pharmacological studies of 20-(S)-camptothecin and 20-(S)-9-nitrocamptothecin as anticancer agents.

Groups of 52 and 29 patients with refractory cancers received either native camptothecin (CPT) or 9-nitrocamptothecin (9NC), respectively, in Phase I clinical trials designed to determine the maximum tolerated dose, toxicity and potential efficacy of orally administered camptothecins. Favorable responses occurred with both compounds (11% after CPT, 24% after 9NC). Although both agents could be taken safely for extended periods, dose limiting toxicities were substantial. Diarrhea was the major clinical problem with CPT, and myelosuppression with 9NC. Both compounds could cause hemorrhagic cystitis. The antitumor activity demonstrated suggests that further investigation of orally administered camptothecin analogs is warranted.

Establishment of human prostate tumor xenografts in nude mice and response to 9-nitrocamptothecin in vivo and in vitro does not correlate with the expression of various apoptosis-regulating proteins.

Flow cytometry and microscopy analyses have demonstrated that 9-nitrocamptothecin (9NC) induces apoptosis in prostate carcinoma LNCaP, DU-145 and PC-3 cells grown in culture or as xenografts. 9NC induces apoptosis regardless of the ability of the cells to induce tumors following xenografting into nude mice. Detection of apoptosis by flow cytometry was preceded or accompanied by increased cell size, loss of nuclear structure and vacuolization, as the tumor regressed, but no visible chromatin fragmentation. This is the first demonstration that 9NC is curative for human prostate carcinoma xenografts in the nude mouse model in the absence of detectable drug-induced toxicity during and after tumor regression. These findings indicate that 9NC may develop into a chemotherapeutic drug for the effective treatment of prostate cancer patients. Further, there was no apparent correlation of the steady-state level of the apoptosis-regulating proteins, Bcl-2, Bcl-XL, Bax and Ich-1, with tumorigenicity of the prostate cells xenografted in nude mice, aggressiveness of tumors grown in nude mice, and induction of apoptosis by 9NC. However, the TIAR protein was present at markedly high levels in all prostate carcinoma cell lines and this may correlate with their susceptibility to 9NC-induced apoptosis.