Real-world benefit of nivolumab in a Canadian non-small-cell lung cancer cohort.

Background: Nivolumab was the first immuno-oncology agent available for the treatment of lung cancer in Canada. In the present study, we evaluated the real-world benefit of nivolumab in Canadian patients with lung cancer. Methods: Patients included in the cohort were identified from a registry of patients treated through expanded access to nivolumab before and after Health Canada approval. Demographics were collected from the application forms. Outcome data for the duration of treatment and survival were collected retrospectively. Results: In contrast to the randomized clinical trial populations, our study cohort included patients who were older (median age: 66 years; range: 36-92 years) and who had an Eastern Cooperative Oncology Group performance status of 2 (8.9%). Despite the poorer-prognosis cohort, median overall survival was 12.0 months, which is comparable to the survival demonstrated in the randomized phase iii trials of nivolumab in lung cancer. Median time to treatment discontinuation was 3.45 months and was similar for all patient subgroups, including poorer-prognosis groups such as those with a performance status of 2, those 75 years of age and older, and those with brain metastases. Conclusions: Nivolumab given in a real-world clinical setting was associated with results similar to those reported in the phase iii clinical trial setting.

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

Methotrexate (MTX-Glu1) exerts its antitumor effects through its potent inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for maintaining the cellular pool of reduced folates. Since the drug-enzyme complex (bound drug) is slowly dissociable, an excess of drug (unbound or free drug) above that required to bind all enzyme sites is required in order to compete with substrate for sites made available by enzyme-drug dissociation. We have examined the role of the polyglutamyl metabolites of MTX-Glu1 containing two to five glutamyl (MTX-Glu2-5) groups in gamma peptide linkage in maintaining an intracellular pool of free drug and in forming slowly dissociable complexes with DHFR. During 24-h incubations of ZR-75-B human breast cancer cells with 2 microM MTX-Glu1, we observed the progressive formation of derivatives with two to five glutamyl groups, which rapidly replaced the parent compound on enzyme binding sites and represented 85% of both unbound and bound intracellular drug at the end of incubation. When cells were then placed in drug-free medium, the rates of disappearance of drug and metabolites from the intracellular bound and free fractions decreased with increasing glutamyl chain length. Over 90% of both bound and free MTX-Glu1 left the cells within 1 h, greater than 90% of MTX-glu2 left within 6 h, and greater than 90% of MTX-Glu3 left the bound and free fractions within 24 h. In contrast, free MTX-Glu4 fell by only 63% and bound by only 23% after 24 h, while free MTX-Glu5 increased by 52% after 6 h in drug-free medium and bound MTX-Glu5 increased threefold after 24 h, as it replaced the other forms of drug bound to DHFR. These results suggested a rapid dissociation of MTX-GLu1 and -Glu2 from the enzyme, and a slower dissociation of the longer chain length derivatives. This conclusion was confirmed by examining the rates at which [3H]MTX-Glu1 through -Glu5 could be replaced on enzyme binding sites by a fivefold or greater excess of unlabeled MTX-Glu1. Bound [3H]MTX-Glu1 and -Glu2 had dissociation t 1/2 of 12 and 30 min, respectively, while -Glu3, -Glu4, and -Glu5 had t 1/2 of 102, 108, and 120 min. These experiments demonstrated that the longer chain polyglutamates have prolonged intracellular retention and can be dissociated less readily than MTX-Glu2 from DHFR, properties likely to make them more efficient DHFR inhibitors than the parent drug and of potential importance in extending the duration of drug action in tumor cells.

Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.

To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 muM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates. These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

We have characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with less than 0.1 microM MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 microM) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 microM) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 microM drug. Two cell lines with resistance to 10 microM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 microM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.

Lack of interference by the unnatural isomer of 5-formyltetrahydrofolate with the effects of the natural isomer in leucovorin preparations.

Leucovorin, a mixture of the natural (6S) and unnatural (6R) diastereomers of 5-formyltetrahydrofolate, is administered clinically to enhance the antitumor activity of 5-fluorouracil. Because the 6R isomer persists at high concentrations in plasma for prolonged periods after iv leucovorin administration (J Clin Oncol 4:685-696, 1986), we have examined it to ascertain whether the 6R isomer could interfere with the cellular effects of the 6S isomer. The 6R compound had a poorer uptake into human CCRF-CEM lymphoblastic cells than the 6S compound, but the 6R compound could competitively inhibit the uptake of the natural isomer as determined in defined buffers. However, the 6R compound failed to interfere with cell growth support and enhancement of 5-fluorouracil cytotoxicity by the 6S isomer in CCRF-CEM cells in tissue culture experiments at concentrations up to 1 mM. Thus, the unnatural isomer of 5-formyltetrahydrofolate present in leucovorin preparations seems unlikely to have clinically relevant consequences.

Prevention of methotrexate cytotoxicity by asparaginase inhibition of methotrexate polyglutamate formation.

Escherichia coli asparaginase (Asnase) pretreatment of Asnase-sensitive L5178Y cells in vitro is thought to antagonize methotrexate (MTX) cytotoxicity through nonspecific inhibition of protein synthesis and MTX uptake. We have reexamined the mechanism of this interaction in view of recent data demonstrating the importance of MTX metabolism to polyglutamate derivatives (MTXPGs) in the cytotoxic effects of the antifolate. After a 3-hr exposure to 0.5 microM MTX, 67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2-5), and cloning efficiency in drug-free medium was only 7% of untreated control. After a 3-hr pretreatment with E. coli Asnase (0.1 unit/ml), [3H]thymidine incorporation dropped by 29%, MTXPG formation during subsequent MTX exposure decreased by more than one-half (MTX-Glu2 unchanged; MTX-Glu3 and 4 decreased to 51.7 and 18.5% of levels achieved in cells not pretreated with Asnase; no MTX-Glu5 formed), and cloning efficiency increased to 71% of untreated control. This effect was not due to decreased MTX uptake into L5178Y cells or to decreased intracellular free L-glutamate or L-glutamine levels. A 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an essential amino acid for cell growth, prior to MTX exposure inhibited [3H]thymidine incorporation by 37%, decreased subsequent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels. Decreased MTXPG formation was responsible for the prevention of MTX cytotoxicity seen after both pretreatments. Unmetabolized MTX rapidly left L5178Y cells after removal of extracellular MTX. Consequently, lower levels of unbound intracellular drug, a prerequisite of drug activity, were maintained in pretreated than in control cells after passage in drug-free medium. Asnase pretreatment protects L5178Y cells from the cytotoxic effects of MTX, possibly through inhibition of cell growth which nonspecifically decreases MTXPG formation.

Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.

Human liver methenyltetrahydrofolate synthetase: improved purification and increased affinity for folate polyglutamate substrates.

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP- and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30 degrees C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.

Identification and characterization of human mitochondrial methenyltetrahydrofolate synthetase activity.

We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa). Studies using purified mitochondrial MTHFS from CA46 human Burkitt lymphoma cells reveal that mitochondrial MTHFS behaves kinetically like the cytoplasmic enzyme with Km values of 4.7, 0.8 and 22 microM respectively for (6R,S)-5-formyltetrahydrofolate monoglutamate, (6S)-5-formyltetrahydrofolate pentaglutamate and ATP. This finding adds to previous observations that various folate-dependent enzymes reside in the mitochondria of eucaryotic cells. Intracellular tetrahydrofolate metabolism is highly compartmentalized and mitochondrial MTHFS activity is necessary for the entry of mitochondrial 5-formyltetrahydrofolate into the mitochondrial folate pool.

Troxacitabine, a novel dioxolane nucleoside analog, has activity in patients with advanced leukemia.

PURPOSE: To investigate the toxicity profile, activity, and pharmacokinetics of a novel L-nucleoside analog, troxacitabine (BCH-4556), in patients with advanced leukemia. PATIENTS AND METHODS: Patients with refractory or relapsed acute myeloid (AML) or lymphocytic (ALL) leukemia, myelodysplastic syndromes (MDS), or chronic myelogenous leukemia in blastic phase (CML-BP). Troxacitabine was given as an intravenous infusion over 30 minutes daily for 5 days. The starting dose was 0.72 mg/m(2)/d (3.6 mg/m(2)/course). Courses were given every 3 to 4 weeks according to toxicity and antileukemic efficacy. The dose was escalated by 50% until grade 2 toxicity was observed, and then by 30% to 35% until the dose-limiting toxicity (DLT) was defined. RESULTS: Forty-two patients (AML: 31 patients; MDS: six patients [five MDS + one CMML]; ALL: four patients; CML-BP: one patient) were treated. Median age was 61 years (range, 23 to 79 years), and 29 patients were males. Stomatitis and hand-foot syndrome were the DLTs. The MTD was defined as 8 mg/m(2)/d. The pharmacokinetic behavior of troxacitabine is linear over the dose range of 0.72 to 10.0 m/m(2). Approximately 69% of troxacitabine was excreted as unchanged drug in the urine. Marrow hypoplasia occurred between days 14 and 28 in 73% of AML patients. Three complete remissions and one partial remission were observed in 30 assessable AML patients. One MDS patient achieved a hematologic improvement. A patient with CML-BP achieved a return to chronic phase disease. CONCLUSION: Troxacitabine has a unique metabolic and pharmacokinetic profile and significant antileukemic activity. DLTs were stomatitis and hand-foot syndrome. Troxacitabine merits further study in hematologic malignancies.

Phase II study of troxacitabine (BCH-4556) in patients with advanced and/or metastatic renal cell carcinoma: a trial of the National Cancer Institute of Canada-Clinical Trials Group.

PURPOSE: A multi-institution phase II study was undertaken by National Cancer Institute of Canada-Clinical Trials Group to evaluate the efficacy and toxicity of intravenous troxacitabine (Troxatyl; Shire Pharmaceuticals Plc, Laval, Quebec, Canada), in patients with renal cell carcinoma. PATIENTS AND METHODS: Between June 1999 and March 2000, 35 patients (24 male) with a mean age of 60 years who had advanced and/or metastatic disease were treated with troxacitabine given as an intravenous infusion over 30 minutes at a dose of 10 mg/m2 intravenously, once every 3 weeks. RESULTS: Of the 33 of 35 patients evaluable for response, there were two confirmed partial responses, 21 patients had stable disease (median duration, 4.4 months), and 10 patients had progressive disease. Eight patients remained stable for more than 6 months, of whom six remain free of progression. The most common drug-related nonhematologic toxicities observed were skin rash (77.1%), hand-foot syndrome (68.6%), alopecia (51.4%), fatigue (51.4%), and nausea (57.1%). Out of a total of 145 cycles of treatment, 98 were given without steroid premedication, whereas 47 cycles were given with steroid premedication. Without premedication, skin rash occurred in 37% of cycles compared with 26% when steroids were given prophylactically. CONCLUSION: Troxacitabine given at a dose of 10 mg/m2 once every 3 weeks was well tolerated in patients with metastatic renal cell cancer, with common toxicities being a moderate to severe granulocytopenia and skin rash. Steroid premedication may reduce the frequency and severity of the skin rash. Our current study suggests that the nucleoside analog troxacitabine may have modest activity against renal cell carcinoma; however, larger studies are required to confirm this.

The new dioxolane, (-)-2'-deoxy-3'-oxacytidine (BCH-4556, troxacitabine), has activity against pancreatic human tumor xenografts.

There is a great need for new therapeutic agents for patients with advanced pancreatic cancer. The new dioxolane analogue troxacitabine was evaluated in two human pancreatic cancer xenograft models. The models used included the Panc-01 and MiaPaCa pancreatic cancer cell lines. Whereas there is certainly no absolute evidence that either of the in vivo models is predictive for clinical activity, there is at least some evidence that they may be helpful in selecting agents for clinical trials in patients with pancreatic cancer. Troxacitabine was administered i.v. to the animals at doses of 10 and 25 mg/kg on a daily x 5 regimen. Gemcitabine was used as a positive control. The end points for the study included tumor growth inhibition (TGI), final weight, and the number of partial and complete tumor responses in the animals. Troxacitabine was highly active against the Panc-01 model (n = 8), with TGI levels of 88.5% and 84.3% at the 10 and 25 mg/kg doses, respectively. The mean final tumor weights for animals given troxacitabine were also significantly smaller (P < 0.001) compared with vehicle controls. At the 10 mg/kg dose, there were three partial tumor shrinkages and one complete tumor shrinkage, whereas at the 25 mg/kg dose, there were three partial tumor shrinkages. Troxacitabine had less activity against the MiaPaCa model (n = 10) and, by traditional response criteria, would be considered inactive, with TGIs of 4% and 22.7% at the 10 and 25 mg/kg dose level, respectively. Of note is that in comparison with gemcitabine, troxacitabine was more efficacious against Panc-01 and was equally active against MiaPaCa. These in vivo results are encouraging and support the prospect of performing Phase II and perhaps Phase III trials with troxacitabine in patients with advanced pancreatic cancer.

Test dose for predicting high-dose methotrexate infusions.

Eighteen evaluable patients were studied to determine whether individual methotrexate (MTX) kinetics, determined by test-dose bolus injection, could be used to predict plasma drug concentrations during and after high-dose infusion. Small nontoxic doses of MTX (10 mg/m2) was given to patients who were followed for 12 to 24 hr and the kinetic data were used to predict subsequent kinetic behavior of moderate- and high-dose methotrexate infusions (150 to 1500 mg/m2 over 12 to 18 hr). After test-dose injection, MTX clearance varied from 36 to 138 ml/min/m2 and decreased with advancing age (r = -0.49, P less than 0.05). MTX clearance varied from 24 to 100 ml/min/m2 after high-doses. Although there was a trend to decreasing clearance with advancing age, this was not as clear as with the test dose (r = -0.42, P greater than 0.05). There was no correlation between MTX clearance and creatinine clearance in this group of patients in whom creatinine clearance varied from 32 to 63 ml/min/m2. When the kinetic parameters derived from the test-dose data were used, accurate predictions could be made of the infusion plateau (r = 0.89, P less than 0.001) and 24-hr (r = 0.92, P less than 0.001) MTX concentrations after high-dose infusions. Our results indicate that test-dose MTX kinetics may serve as a guide to dose modification of MTX infusions in some high-risk patients.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Biochemical and Molecular Studies of Human Methenyltetrahydrofolate Synthetase.

5-FormylH(4)folate is administered clinically under the name Leucovorintrade mark in association with the antineoplastic agent 5-fluorouracil (5-FU) to enhance the cytotoxic effects of 5-FU. The combination has been shown to be superior to 5-FU alone in the treatment of patients with metastatic colorectal carcinoma. Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the transformation of 5-formyltetrahydrofolate to methenylH(4)folate, which is the obligatory initial metabolic step prior to the intracellular conversion of 5-formylH(4)folate to other reduced folates and the increase in intracellular folate pools required for 5-FU potentiation. In the following paper, we will summarize results of biochemical and molecular studies of human MTHFS.

Role of leucovorin dosing and administration schedule.

Leucovorin (LV) is commonly administered in association with 5-fluorouracil (5-FU) to enhance its cytotoxic effects. In this paper, the cellular and clinical pharmacology of 5-FU potentiation by LV are reviewed, and the dosing and administration schedules are discussed in relation to reported clinical trials. In vitro experimental data suggest that prolonged cellular exposures to relatively low LV concentrations simultaneously with prolonged 5-FU administration are the optimal conditions to enhance 5-FU efficacy. Clinical studies of 5-FU/LV in metastatic colorectal carcinoma have established that 5-day bolus 5-FU with low-dose bolus LV injections yield therapeutic benefits equivalent to those obtained with intravenous bolus schedules using higher doses of LV. It remains to be determined, however, if bolus administration schedules are the optimal clinical treatment regimens. Infusional 5-FU/LV regimens appear to be a strategy worthy of further clinical investigation.

The importance of dose scheduling with mitoxantrone, 5-fluorouracil and leucovorin in metastatic breast cancer.

We have studied a mitoxantrone, 5-fluorouracil (5-FU) and leucovorin chemotherapy regimen in metastatic breast cancer. 8 patients received mitoxantrone 10 mg/m2 on day 1, leucovorin 200 mg/m2 and 5-FU 300 mg/m2 on days 1-5 by intravenous bolus every 28 days in a pilot study. Grades 3-4 granulocytopenia followed 55% of the courses, with 2 patients admitted for febrile neutropenia. Only a 29% objective response rate was seen in a subsequent phase II trial using reduced mitoxantrone doses. Comparison with other trials suggested that 5-day bolus 5-FU administration adversely affects the combination's therapeutic index.

Leucovorin rescue of human cancer and bone marrow cells following edatrexate or methotrexate.

We have examined the cytotoxic activities of edatrexate (EDX) and methotrexate (MTX) and their reversal by leucovorin in nine human cancer cell lines and in human bone marrow CFU-GM cells. EDX was 3.7- to 123-fold more toxic than MTX against the cancer cell lines and 25-fold against the bone marrow cells. Lower EDX concentrates generally were needed to inhibit cancer cell growth relative to bone marrow cells, however, whereas bone marrow and cancer cell growth were more often susceptible to the same MTX concentrations. The new antifolate was metabolized to long-chain polyglutamates to a greater extent than MTX in seven cell lines. Leucovorin at 0.2 microM rescued two breast cancer and two non-small cell lung cancer cell lines to a lesser extent following EDX than MTX, but significant rescue was observed in two head and neck cancer cell lines that formed large amounts of polyglutamates. These cell lines also accumulated reduced folates to a greater extent than the other cell lines following leucovorin exposure. Leucovorin rescued bone marrow cells following MTX but only partially following the highest EDX concentrations. EDX may enjoy a better therapeutic index than MTX against some cancer cell lines relative to bone marrow precursor cells, especially after leucovorin rescue.

The palliative benefit of irinotecan in 5-fluorouracil-refractory colorectal cancer: its prospective evaluation by a Multicenter Canadian Trial.

Most patients with colorectal cancer (CRC) who have failed initial 5-fluorouracil (5-FU) chemotherapy have worsening of disease-related symptoms (DRS) and quality of life (QOL). Irinotecan has a reported response rate of 10%-20% in such patients. The aim of this phase II trial was to prospectively determine the palliative benefit of irinotecan utilizing DRS as primary endpoints of response. Patients had advanced CRC refractory to 5-FU with at least 1 DRS defined as (1) Karnofsky performance status (KPS) 60%-80%, (2) baseline analgesic use > or = 10 mg morphine/day (or equivalent), or (3) disease-related pain score > 1 cm on a 10-cm linear analogue self-assessment (LASA) scale. Patients received irinotecan 125 mg/m2 weekly for 4 weeks on an every-6-weeks schedule. The primary endpoint was palliative response defined as > or = 50% decrease in pain score or analgesic usage, or 10% increase in KPS, from baseline for 4 weeks. QOL was assessed by the European Organization for Research and Treatment of Cancer Quality-of-Life Questionnaire Core 30 (EORTC QLQ-C30) version 2 instrument. A total of 65 patients were entered onto the study. Median baseline parameters were KPS 70%, analgesic score 11 mg/day, and pain score 2.4 cm. A palliative response was achieved in 27 patients (42%), improvement in pain score predominated. LASA and EORTC QLQ-C30 instruments showed parallel changes in DRS. The radiological response rate was 11% (complete responses and partial responses, n = 46); 23 patients achieved stable disease. Median overall survival was 7.2 months. Irinotecan provides a rate of palliative benefit higher than the radiological response rate. Patients-oriented palliative endpoints can be useful in assessing the benefit of agents in early-phase clinical trials.

5-Fluorouracil-metronidazole combination therapy in metastatic colorectal cancer. Clinical, pharmacokinetic and in vitro cytotoxicity studies.

We have investigated the role of metronidazole (MND) combined with 5-fluorouracil (5-FU) in the treatment of metastatic colorectal cancer. MND (750 mg/m2) was administered i.v. 1 h before 5-FU (600 mg/m2) i.v., daily for 5 consecutive days. Treatment was repeated every 4 weeks until disease progression or prohibitive toxicity occurred. Of the 27 patients entered in the study, 4 (15%) had an objective complete or partial response lasting an average of 7 months. 5-FU toxicity was greatly enhanced by the administration of MND, however, 74% of patients having granulocytopenia (less than 1500/microliter). We investigated the possible mechanisms underlying this enhanced 5-FU toxicity by examining whether MND modified 5-FU pharmacokinetics or whether the two drugs had a synergistic effect in vitro against the HCT-8 colon cancer cell line. While the in vitro studies failed to reveal any synergism between 5-FU and MND, pharmacokinetic evaluation revealed that 5-FU clearance was significantly reduced (26.9%, P less than 0.001) by prior MND administration. MND reduces 5-FU's therapeutic index in the treatment of colorectal cancer by impairing its clearance, which leads to increased toxicity without enhanced therapeutic efficacy.