Pharmacokinetics of concomitant cisplatin and paclitaxel administered by hyperthermic intraperitoneal chemotherapy to patients with peritoneal carcinomatosis from epithelial ovarian cancer.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is advised as a treatment option for epithelial ovarian cancer (EOC) with peritoneal carcinomatosis. This study was designed to define the pharmacokinetics of cisplatin (CDDP) and paclitaxel (PTX) administered together during HIPEC. METHODS: Thirteen women with EOC underwent cytoreductive surgery (CRS) and HIPEC, with CDDP and PTX. Blood, peritoneal perfusate and tissue samples were harvested to determine drug exposure by high-performance liquid chromatography and matrix-assisted laser desorption ionization imaging mass spectrometry (IMS). RESULTS: The mean maximum concentrations of CDDP and PTX in perfusate were, respectively, 24.8±10.4 µg ml(-1) and 69.8±14.3 µg ml(-1); in plasma were 1.87±0.4 µg ml(-1) and 0.055±0.009 µg ml(-1). The mean concentrations of CDDP and PTX in peritoneum at the end of HIPEC were 23.3±8.0 µg g(-1) and 30.1±18.3 µg(-1)g(-1), respectively. The penetration of PTX into the peritoneal wall, determined by IMS, was about 0.5 mm. Grade 3-4 surgical complications were recorded in four patients, five patients presented grade 3 and two patients presented grade 4 hematological complications. CONCLUSIONS: HIPEC with CDDP and PTX after CRS is feasible with acceptable morbidity and has a favorable pharmacokinetic profile: high drug concentrations are achieved in peritoneal tissue with low systemic exposure. Larger studies are needed to demonstrate its efficacy in patients with microscopic postsurgical residual tumours in the peritoneal cavity.

Phase I study of the antipurine antifolate lometrexol (DDATHF) with folinic acid rescue.

Lometrexol (5,10-dideazatetrahydrofolic acid) is a new antifolate that is highly selective in inhibiting the key enzyme of purine synthesis glycinamide ribonucleotide formyltransferase. The most promising preclinical features of lometrexol in animal models were its significant activity against a broad panel of solid tumors, the schedule dependency of its antitumor activity, and the availability of a rescue regimen with folinic or folic acid. In the present study, lometrexol was first given daily for 3 consecutive days, repeated every 4 weeks (part I). The occurrence of delayed myelotoxicity prompted the development of a rescue regimen with lometrexol given in a single dose on day 1, followed by oral folinic acid, 15 mg four times a day, from day 3 to day 5 (part II). Longer time intervals between administration of lometrexol and start of rescue were then evaluated (part III), and in the last part of the study (part IV), the maximum tolerated dose of single intermittent doses of lometrexol with folinic acid given from day 7 to day 9 was established. Sixty adult patients entered the study. In part I, the highest daily dose that could be safely given was 4 mg/m2, for a total dose of 12 mg/m2. Cumulative early stomatitis and delayed thrombocytopenia were dose limiting. The use of oral folinic acid made it possible to escalate the dose up to 60 mg/m2, and the maximum tolerated dose was reached at this dose when folinic was given from day 7 to day 9, with anemia being the dose-limiting toxicity. A shorter time interval between lometrexol and folinic acid administrations (from day 5 to day 7) is recommended for Phase II evaluations to optimize the antitumor effect. Anemia was normochromic and macrocytic, possibly due to a deficiency of folic acid. One partial response of 8 months' duration was reported in a patient with epithelial cancer of the ovary, relapsing after cisplatin and alkylating agents. The use of folic acid as rescue, proposed on the basis of experimental data and pharmacological considerations, has also allowed the repeated administration of lometrexol at doses higher than in the previous studies. The advantages of rescue with folinic acid over supplementation with folic acid, however, are difficult to define.

Role of membrane folate-binding protein in the cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cell lines in vitro.

Lometrexol (5,10-dideazatetrahydrofolic acid; DDATHF), is a specific inhibitor of glycinamideribonucleosyl (GAR) transformylase with anti-tumour activity in murine and human carcinomas. The cytotoxicity activity of DDATHF was evaluated in vitro in NIH/3T3 cells transfected with human alpha-folate-binding protein (FBP) complementary DNA to examine the role of the receptor. In FBP-transfected NIH/3T3 (FBP-tNIH/3T3) cells, which internalised about three times more 5-methyltetrahydrofolic acid than the mock-transfected cells, the cytotoxtic potential of DDATHF showed a clear increase. Subsequently, we analysed four ovarian carcinoma cell lines (OVCAR3, IGROV1, SKOV3, and SW626) expressing different amounts of FBP. Cells were conditioned to grow in medium depleted of folic acid then tested by MOv18 and folic acid binding. Only SKOV3 and SW626 cells grown in folic acid-depleted medium showed increased FBP expression, about 3- and 8-fold respectively. The cytotoxic potential of DDATHF was evaluated by a standard clonogenic assay. In a medium containing 2.27 microM folic acid the DDATHF IC50 values were 50 nm on OVCAR3, 500 nM on SW626 and 1000 nM on IGROV1. In folic acid-free medium IC50 values were 2 nM on OVCAR3 and Sw626 and 40 nM on IGROV1. Only on SKOV3 cells was DDATHF cytotoxicity the same regardless of the amount of folic acid in the medium (IC50 8 nM). Thus, DDATHF did not inhibit the growth of IGROV1 cells depleted of folic acid after stripping FBP with phosphatidylinositol-phospholipase C, even at a dose toxic for cells constitutively expressing FBP. Although FBP expression is certainly one of the parameters affecting drug toxicity, taken alone it is not a sufficiently reliable predictor of cancer cell sensitivity to DDATHF.

O6-methylguanine and temozolomide can reverse the resistance to chloroethylnitrosoureas of a mouse L1210 leukemia.

Using L1210 and a subline resistance to chloroethylnitrosoureas (L1210/BCNU), we found that the resistance to 1-(2-chloroethyl)-1-nitrosourea (CNU) or to diethyl-1-3-(2-chloroethyl)-3-nitrosoureido ethyl phosphonate (fotemustine) can be reversed by a pretreatment with O6-methyl Guanine (O6-mGua) or temozolomide. In L1210/BCNU but not in L1210 the pretreatment with O6mGua caused an increased peak level of CNU-induced DNA-interstrand crosslinks. We then evaluated whether the resistance to BCNU could be counteracted in vivo by i.p. O6mGua treatment of L1210/BCNU bearing mice. The results were negative due to the fact that O6mGua, which was not toxic when given alone, caused a high toxicity when associated with BCNU.

Flavone acetic acid pharmacokinetics in nude mice.

The pharmacokinetics of flavone acetic acid (FAA) were comparatively studied in Balb-c mice and in immunocompetent nude mice treated with i.v. doses of 100 and 300 mg/kg. In both strains of mice, FAA kinetics appear to be dose-dependent, since the AUC values increased disproportionally to the dose. FAA protein binding and the pattern of distribution were similar in Balb-c and nude mice, the highest drug concentrations being found in liver and small intestine and the lowest in brain. Renal excretion appears to be the major route of FAA elimination in both strains, accounting for about 75% of the total drug administered after a dose of 100 mg/kg and 50-60% after a dose of 300 mg/kg. We conclude that FAA pharmacokinetics are comparable in Balb-c and nude mice so that the previously investigated FAA dosage schedules in inbred mice can also be employed in nude mice bearing human tumors.

Increase in topoisomerase-II-mediated DNA breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate.

Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.

Characterization of a novel mouse reticular cell sarcoma M5076 subline resistant to cisplatin.

A novel murine tumor resistant to cis-diamminedichloroplatinum (cisplatin, DDP) was obtained (M5/DDP) after 22 passages in which mice bearing the ovarian reticular cell sarcoma M5076 (M5) were treated with DDP. Although DDP conserved some inhibitory activity on growth of M5/DDP, it was much less effective than on M5. Treatment with DDP did not prolong the survival time of mice with M5/DDP, whereas it markedly prolonged survival of M5-bearing mice. M5 and M5/DDP tumors shared many biological and biochemical features. They were similar histologically, they metastasized reproducibly to the liver and were poorly immunogenic. Their growth rates were comparable; their DNA index, percentage of cells in S phase and intra-cellular glutathione content were also similar. In both tumors, DDP caused an accumulation of cells in S late-G2-M within 24 hr after drug treatment. However, this was efficiently reversed in M5/DDP, whereas it worsened and persisted longer in M5. Cross-resistance was observed between DDP and its analogues carboplatin and iproplatin, but tetraplatin retained marginal activity on M5/DDP tumor. Several alkylating agents tested [L-phenyalanine mustard (L-PAM); cyclophosphamide (CTX); chlorambucil (CLB); 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and dacarbazine (DTIC)] were not totally cross-resistant to DDP, but showed greater activity on M5 than on M5/DDP. Other non-alkylating anti-neoplastic drugs showed a similar degree of activity on M5 and M5/DDP. 5-Aza-2'-deoxycytidine (Aza-d-Cyd) was very effective on both tumors, etoposide (VP-16) and cytosine arabinoside (Ara-C) had no activity and Adriamycin (ADR) was weakly effective.

Antitumor activity of the novel nitrosourea S10036 in rodent tumors.

The activity of the novel anticancer agent diethyl 1-3-(chloroethyl)-3-nitrosoureido ethyl phosphonate (S10036) was investigated on several rodent tumors. S10036 showed a good efficacy, comparable to that of the anticancer agent BCNU, against i.p transplanted P388 and L1210 leukemias. S10036 was very effective against the primary tumor and metastases of i.m transplanted M5076 reticular cell sarcoma of the mouse and against subline A of the Walker carcinoma of the rat. It was inactive against rodent tumors resistant to BCNU such as L1210/BCNU, ICIG-Ci4 murine fibrosarcoma and the Walker carcinoma subline B in the rat.

Human ovarian tumors in primary culture: growth, characterization and initial evaluation of the response to cis platinum treatment in vitro.

A novel cell culture system is reported for the growth of ovarian tumors. Two approaches were developed to isolate tumor cells, one for ovarian carcinomas and the other for benign cystomas or borderline cystadenomas, which yield virtually pure tumor-cell clusters. The plating efficiency exceeded 10% in approximately 80% of the processed surgical specimens. Cells grown in a newly developed KOV medium (a modification of MCDB 151 supplemented with 6 defined growth factors and a moderate amount of FBS) had an average growth rate of 0.23 population doublings/day. Primary tumor-derived cultures, including those derived from cystomas, were analyzed by flow cytometry demonstrating a DNA heteroploid content in 55% of the cases. The neoplastic origin of the cells in culture was further confirmed by 3 monoclonal antibodies (OC125; MOv2; MOv19) with high specificity against epithelial ovarian malignancies. Cultures were tested with cis-DDP to determine their suitability for pharmacological studies. Exposure to the drug (from 10 to 80 microM for 1 hr) resulted in variable cell-killing responses, and the prominent effect on cell-cycle progression in primary cultures was a prolonged arrest in S phase. The formation and persistence of DNA-ISC caused by an exposure to 40 microM cis-DDP for 1 hr was studied by alkaline elution in 6 different tumor-derived cultures. DNA-ISC equivalents were highest between 9 and 24 hr after treatment and were repaired only to a limited extent within 48 hr of recovery time. The present study confirms the usefulness of this culture system for pharmacological studies of active chemotherapeutic agents against human ovarian tumors.

Accumulation of DNA strand breaks in cells exposed to methotrexate or N10-propargyl-5,8-dideazafolic acid.

N10-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly inhibits thymidylate synthase and which is now under early clinical investigation, was compared with methotrexate (MTX) for its antiproliferative activity and mode of action on M14 human melanoma cell line and NIH/3T3 murine fibroblasts transfected with human c-Ha-ras oncogene (NIH/3T3R). CB 3717 was as active as MTX on both cell lines in inhibiting colony formation, but 20-100 times less potent. After 24 h of exposure both drugs caused an accumulation of cells in the G1 phase of the cell cycle, probably because of inhibition of DNA synthesis and blockage at the G1-S boundary. In NIH/3T3R treated for 16 h with 2 microM MTX or 200 microM CB 3717, we found DNA single-strand breaks amounting to approximately 130 and 140 rad equivalents, respectively, and a considerable number of DNA double-strand breaks, far more than expected if they had been the result of the proximity of single-strand breaks on the two complementary DNA strands. No DNA-protein cross-links were detected. When cells were incubated in drug-free medium for 8 h, there was a further accumulation of single-strand breaks, possibly due to the effects of the drug retained intracellularly as polyglutamyl derivative. Simultaneous treatment with 1.77 microM cycloheximide prevented DNA damage produced by both drugs. Thymidine (10 microM), renewed in the culture medium every 24 h, also prevented DNA damage and cytotoxicity. Since after 16 h treatment with MTX or CB 3717 cells were completely viable, as assessed by [3H]thymidine release, trypan blue exclusion test, and 51Cr release, DNA damage appears to be an early event preceding cell death and may be a feature of the killing ability of the drugs. The involvement of a protein in the formation of DNA breaks is suggested by the fact that when protein synthesis was inhibited with cycloheximide DNA damage was no longer seen.

In vivo studies with the novel anticancer agent mitozolomide (NSC 353451) on Lewis lung carcinoma.

Mitozolomide is one of the most effective drugs against Lewis lung carcinoma in the mouse. Two IP doses of 40 mg/kg (days 6 and 15 after IM transplantation of 3LL) or four doses of 20 mg/kg given at various intervals (starting from day 6) increased survival time by 100%. A single IP dose of 80 mg/kg was toxic, and 10 mg/kg was ineffective even when this dose was given on eight occasions. The pharmacokinetics of mitozolomide was investigated in 3LL-bearing mice by HPLC assay. Peak drug levels were achieved in tumor 15 min after IP treatment, after which they declined according to first-order kinetics, with a half-life of 80-100 min (the same as in plasma). No dose-dependent kinetics was observed. Flow cytometry studies showed an accumulation of 3LL cells in G2M 24 h after drug treatment. This cell cycle perturbation was reversed 96 h after the inactive dose of 10 mg/kg, but not after the effective dose of 40 mg/kg.

Activity and pharmacokinetics of teniposide in Lewis lung carcinoma-bearing mice.

The antitumor activity of teniposide (VM26) was investigated in Lewis lung carcinoma (3LL) of the mouse after a single dose of 20 mg/kg iv (given 8 or 14 days after tumor transplant) or after three doses of 6.5 mg/kg iv (given on Days 8, 11, and 14 after tumor transplant) (total dose, 19.5 mg/kg). The single dose resulted in only 25% primary tumor reduction but had marked antimetastatic activity. The repeated doses (6.5 mg/kg x 3) were much more effective, with 85% primary tumor reduction and the apparent disappearance of all metastatic deposits. The pharmacokinetics of VM26 was investigated in 3LL-bearing mice by a high-performance liquid chromatographic assay. At both of the above doses VM26 disappeared from mouse plasma biphasically, with an elimination half-life of about 70 mins. The concentrations in metastases were higher than in primary tumor, where very low levels were found. The highest VM26 levels were found in liver, small intestine, and kidney, and the lowest levels were found in the brain. Excretion of VM26 in urine amounted to less than 5% of the dose.

Pharmacokinetic approach to in vitro testing of ovarian cancer cell sensitivity.

Cell populations obtained from ovarian cancer specimens were seeded in primary culture and morphologically identified as cancer cells. Methotrexate, cytosine arabinoside, 5-fluorouracil, antinomycin D, melphalan, and adriamycin were added to the culture medium at different concentrations and for various periods of time. The results are discussed in relation to the pharmacokinetic availability of drugs in the plasma compartment of patients treated by different therapuetic regimens. Totally inactive drugs can be identified by comparing plasma levels with active concentrations while for drugs active in vitro at concentrations in the range of pharmacokinetic levels, the percentage of responders among patients might be explained by the intrinsic variability of cancer cells.