Photoradiation therapy. II. Cure of animal tumors with hematoporphyrin and light.

Exposure of mouse and rat tumors of various types to more than 600 nm light 24 or 48 hours after an injection of hematoporphyrin resulted in a substantial number of long-term cures. Since hematoporphyrin is preferentially retained in tumor tissue, selective tumor destruction could be obtained. Light penetration studies and the high efficiency of this technique indicated its applicability even to certain deep-seated human tumors.

Antitumor activity and clinical pharmacology of sulofenur in ovarian cancer.

BACKGROUND: Sulofenur is a diarylsulfonylurea with demonstrated antitumor activity in patients with advanced epithelial ovarian cancer refractory to standard chemotherapy. The dose-limiting toxic effects observed in phase I clinical trials have been anemia and methemoglobinemia, resulting in cyanosis. PURPOSE: The purposes of this study were to further define the response rate, toxic effects, and pharmacokinetics and pharmacodynamics of sulofenur in patients with advanced ovarian cancer. METHODS: We conducted a phase II trial of sulofenur at a dose of 800 mg/m2 per day in 35 patients with stage III or IV ovarian cancer refractory to standard chemotherapy. Pharmacokinetics and pharmacodynamics were analyzed by comparing sulofenur parent and metabolite plasma levels with methemoglobin levels. RESULTS: Partial responses lasting 6.5-18 weeks occurred in four (15%; 95% confidence interval = 4%-35%) of the 26 patients assessable for response. In addition, 42% (11) of the assessable patients had prolonged stable disease (median, 20 weeks). The first nine patients received sulofenur as a daily oral dose for 14 days, with a 21-day treatment cycle. However, they developed substantial anemia and methemoglobinemia. As a result, the next 26 patients received sulofenur daily for 5 days followed by 2 days of rest for 3 consecutive weeks, with a 28-day treatment cycle (5/2-day schedule). Preclinical models predicted that 2 days of rest would decrease toxicity while maintaining antitumor activity. Patients treated with the 5/2-day schedule had relatively less severe anemia and methemoglobinemia and needed fewer red blood cell transfusions (31% versus 78% of patients), but 31% still required dose reductions because of these toxic effects. The hydroxy and keto metabolites of sulofenur had prolonged plasma half-lives relative to the parent compound, and the difference was statistically significant. In addition, the correlations of metabolite concentrations with methemoglobin levels were higher than the correlation of sulofenur concentrations with methemoglobin levels, and those differences were statistically significant. CONCLUSION: We conclude that sulofenur has modest clinical activity in heavily pretreated patients with ovarian cancer. IMPLICATIONS: The toxic effects of anemia and methemoglobinemia may limit the ultimate clinical utility of diarylsulfonylureas until less toxic derivatives with alternate metabolic pathways can be identified.

Effects of allopurinol on the therapeutic efficacy of methotrexate.

The antitumor effects of methotrexate against early leukemia L1210 were partially reversed by the coadministration of allopurinol in vivo, even though allopurinol did not alter the growth-inhibitory effects of methotrexate against L1210 cells in culture. These data suggest that this alteration in antitumor activity results from a decreased catabolism of preformed systemic purines by allopurinol, a potent inhibitor of xanthine oxidase. On the other hand, the therapeutic effect of methotrexate against the P288 leukemia was not significantly altered by allopurinol did not significantly alter the toxicity of methotrexate that, in the mouse, the antileukemic effects of methotrexate are more related to a purineless rather than a thymineless death.

Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine.

Continuous exposure to inhibitory concentrations of methotrexate produces distinct rates of steady-state growth of murine leukemia L1210 and human leukemia CCRF-CEM cells in culture. Addition of thymidine to the medium produces reversal (6 to 40%) of this steady-state growth rate inhibition. This study utilized combinations of methotrexate and thymidine for an evaluation of the accompanying relationship between steady-state growth rate and changes in the ribo- and deoxyribonucleoside triphosphate pools. In L1210 cells exposed to methotrexate alone, the deoxythymidine 5'-phosphate (dTTP) pools decreased, whereas deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and deoxycytidine 5'-triphosphate (dCTP) remained relatively constant up to 70% inhibition of growth rate, with dCTP at a constant 112% of controls. The corresponding ribonucleoside triphosphates decreased only slightly. With the combination of methotrexate and thymidine resulting in up to 40% inhibition of growth rate, there was also a decrease in the dTTP pool while the other deoxyribonucleoside triphosphates remained relatively constant, and the corresponding ribonucleoside triphosphates again decreased only slightly. The dCTP pool was reduced to a constant 42% of control comparable to that produced by thymidine alone. With greater than 40% (with thymidine) or 70% (without thymidine) inhibition of growth rate, all pools decreased, but only dTTP was substantially reduced in proportion to the growth rate inhibition caused by methotrexate. The dTTP pool became depleted in spite of the presence of exogenous thymidine. Evaluation of CCRF-CEM cells indicated that inhibition of growth rate and nucleotide pool perturbations by methotrexate were similar to those observed in L1210 cells. However, in the presence of thymidine, inhibition of growth rate appeared related to decreased pools of dCTP, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate, rather than dTTP as was observed for L1210 cells. Hence, mammalian cells were capable of responding in a differential fashion to pharmacological perturbations, and this capacity may play a role in determining therapeutic selectivity. Since the ribonucleoside triphosphate decreases were slight and relatively uniform during methotrexate-induced perturbations, the deoxyribonucleoside triphosphate pools appear to be more directly related to inhibition of growth rate. The results are consistent with the concept that slight imbalances in the deoxyribonucleoside triphosphate pools dramatically inhibit DNA synthesis, as mediated through their interaction with DNA polymerase.

Isolation of a DNA polymerase alpha-associated regulatory protein from calf thymus.

A regulatory protein for DNA polymerase alpha, responsive to noncomplementary deoxyribonucleoside triphosphates, has been isolated from calf thymus. The regulatory protein was separated from DNA polymerase alpha using Affi-Gel Blue and gel filtration. The regulatory protein had a molecular weight of approximately 70,000 as determined by gel filtration, and its activity was nondialyzable, heat labile, and abolished by pronase treatment. In the presence of regulatory protein, DNA polymerase alpha activity, measured by using polydeoxyadenylate-oligodeoxythymidylate as template primer, was inhibited by 2'-deoxyguanosine 5'-triphosphate in a parabolic-competitive fashion [Ki = 15 +/- 1 (S.E.) microM] and by 2'-deoxycytidine 5'-triphosphate in a linear-competitive manner (Ki = 162 +/- 23 microM). Neither the four natural ribonucleoside triphosphates nor 2'-deoxyadenosine 5'-triphosphate inhibited the DNA polymerase-regulatory protein system to any significant extent. The regulatory protein by itself had no effect on either DNA polymerase alpha activity or the Km for template primer. These results indicate that deoxyribonucleoside triphosphate pools may be involved in the regulation of cellular DNA synthesis through a direct effect on DNA polymerization.

Comparison of the cellular pharmacokinetics and toxicity of 2',2'-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine.

2',2'-Difluorodeoxycytidine (dFdC) is a new deoxycytidine analogue with good activity against human leukemic cell lines and murine solid tumors, while the activity of 1-beta-D-arabinofuranosylcytosine (ara-C) is established in experimental systems and for the treatment of human adult leukemia. This study compared the cellular metabolism and cytotoxic properties of dFdC and ara-C in Chinese hamster ovary cells. In wild-type cells, dFdC was significantly more cytotoxic than ara-C after both 4- and 18-h incubations. The 5'-triphosphate of dFdC (dFdCTP) was the major cellular metabolite (85-90%), reaching cellular concentrations up to 20-fold greater than those observed for ara-C 5'-triphosphate at equimolar concentrations of the parent drug. A deoxycytidine kinase-deficient mutant neither accumulated dFdCTP nor showed any cytotoxic response up to drug concentrations of 100 microM. The cytotoxicity of dFdC could be competitively reversed by deoxycytidine further suggesting that dFdC, like ara-C, required phosphorylation by deoxycytidine kinase for biological activity. Several explanations for the different cellular accumulation of the drug triphosphates were established: (a) nucleoside transport studies demonstrated that the membrane permeation of dFdC was 65% more rapid than that of ara-C; (b) deoxycytidine kinase had a higher affinity for dFdC (Km = 3.6 microM) than for ara-C (Km = 8.8 microM), while the Km for deoxycytidine was 1.4 microM; (c) the elimination of intracellular dFdCTP was biphasic with t1/2 alpha = 3.9 and t1/2 beta greater than 16 h while the degradation of ara-CTP was monophasic and significantly faster (t1/2 = 0.7 h). The comparatively long half-life of dFdCTP was related to the prolonged inhibition of DNA synthesis after removal of exogenous nucleoside. Together these factors contribute to the more potent cytotoxicity of dFdC compared with ara-C.

Potentiation by guanine nucleosides of the growth-inhibitory effects of adenosine analogs on L1210 and sarcoma 180 cells in culture.

The growth-inhibitory effect of 6-methylmercaptopurine riboside (MMPR) against leukemia L1210 cells in culture was dramatically potentiated by the addition of guanine nucleosides to the medium. In the presence of either deoxyguanosine or guanosine, the concentration of MMPR that caused 50% inhibition of growth was 35 times lower than in the absence of these nucleosides. Similar potentiation was also observed against Sarcoma 180 cells in culture by guanosine. The metabolic basis of this synergism was approached in a study of the incorporation of [14C]glycine into 5'-phosphoribosyl-N-formylglycinamide in Sarcoma 180 cells. The results show that the site of inhibition resulting in synergism is an early step in purine biosynthesis, probably phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14). In the L1210 cell system, the addition of hypoxanthine to the medium prevented the potentiation of MMPR by guanine nucleosides supporting the conclusion that the site of the synergistic interaction involves purine biosynthesis de novo. While hypoxanthine partially reversed the growth-inhibitory effects of MMPR, an even higher degree of protection was observed in the presence of both uridine and hypoxanthine, suggesting that MMPR may have additional sites of action concerned with pyrimidine metabolism.

Action of 2',2'-difluorodeoxycytidine on DNA synthesis.

The action of the new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) on DNA synthesis was investigated in whole cells and in vitro assay systems with purified DNA polymerases. DNA synthesis in human lymphoblastoid CEM cells was inhibited by dFdC in a concentration-dependent manner that could not be reversed by exogenous deoxynucleosides. The analogue was incorporated into cellular DNA; most of the incorporated dFdC 5'-monophosphate (dFdCMP) residues were in internucleotide linkage. In vitro DNA primer extension assays demonstrated that dFdC 5'-triphosphate (dFdCTP) competed with deoxycytidine triphosphate for incorporation into the C sites of the growing DNA strand. The ratios of the apparent Km values for the incorporation of dFdCTP and dCTP into a C site of M13mp19 DNA were 21.8 and 22.9 for DNA polymerases alpha and epsilon, respectively. The apparent Ki values of dFdCTP were 11.2 microM for DNA polymerase alpha and 14.4 microM for polymerase epsilon. After dFdCMP incorporation, the primer was extended by one deoxynucleotide before a major pause in the polymerization process was observed. This was in contrast to the action of arabinosylcytosine 5'-triphosphate, which caused both DNA polymerases alpha and epsilon to pause at the site of incorporation. The 3'----5' exonuclease activity of DNA polymerase epsilon was essentially unable to excise nucleotides from DNA containing dFdCMP at either the 3'-end or at an internal position, whereas arabinosylcytosine monophosphate was removed from the 3'-terminus at 37% the rate for deoxynucleotides. The cytotoxic activity of dFdC was strongly correlated with the amount of dFdCMP incorporated into cellular DNA. Our results demonstrate qualitative and quantitative differences in the molecular actions of dFdC and arabinosylcytosine on DNA metabolism, but are consistent with an important role for such incorporation in the toxicity of dFdC.

Kinetics of N-[(4-chlorophenyl)amino]carbonyl-2,3-dihydro-1H-indene-5-sulfonamide (LY186641) in humans.

The metabolism and disposition of LY186641, a diarylsulfonylurea with antineoplastic activity identified in preclinical tests, was determined in 21 patients who received 23 courses of orally administered drug. A linear correlation was found between the dose of drug administered and LY186641 peak plasma concentrations and LY186641 area under the curve measurements. Clearance (135 +/- 36 ml/h/m2), terminal half-life (31 +/- 11 h), and volume of distribution (10.2 +/- 2.8 liters) were independent of drug dose. No LY186641 was excreted in urine. Hydroxy and keto metabolites of LY186641 were identified in plasma and urine samples. Urinary excretion of these metabolites during the initial 48 h following drug administration accounted for 20% of LY186641 disposition. Plasma half-life of the hydroxy and keto metabolites was longer than that of parent drug (3.3 and 3.1 days, respectively). Plasma concentrations of parent drug correlated with the presence of methemoglobinemia, the dose-limiting toxicity found with LY186641.

Antitumor activity of cytidine dialdehyde and its effects on nucleotide and nucleic acid synthesis.

Cytidine dialdehyde inhibited the growth of leukemia L1210 cells in culture at a 50% inhibitory concentration of 3.5 X 10(-5) M and, when administered i.p. at 200 mg/kg daily for 5 days, increased the mean survival of L1210 tumor-bearing mice by up to 171%. Given by the s.c. or i.v. routes, the compound was ineffective. The ethanol adduct of cytidine dialdehyde, although inactive in cell culture, increased the mean survival of L1210 tumor-bearing mice by up to 225% when administered i.p. but was inactive upon s.c. administration. Exposure of L1210 cells in culture for 25 hr to cytidine dialdehyde at the 50% inhibitory concentration increased the ribonucleoside di- and triphosphate pools, slightly increased deoxyadenosine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate pools, and caused a pronounced increase in the deoxycytidine triphosphate pool. As determined by the rate of pyrimidine precursor incorporation into nucleic acids, this concentration of drug showed no effect on RNA synthesis but caused a reduction in DNA synthesis to 53% of control. Exposure of L1210 cells for 3 to 6 hr to 10(-4) M cytidine dialdehyde, a concentration which inhibits growth completely, effected an increase in the ribonucleoside di- and triphosphate pools and a rapid decrease of the deoxythymidine triphosphate pool. The deoxycytidine triphosphate and deoxyguanosine triphosphate pools decreased more slowly, and the deoxyadenosine triphosphate pool remained slightly elevated. Analysis of the rate of substrate incorporation into nucleic acids showed that this concentration of drug produced an 80% decrease in RNA synthesis and a 75% decrease in DNA synthesis 3 hr after drug exposure. These results suggest that the mechanism of action of cytidine dialdehyde may be due to its initial interference with DNA synthesis followed by a generalized inhibition of DNA, RNA, and protein synthesis at cytotoxic concentrations.

Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a mechanism of self-potentiation.

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine).

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.

N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, a novel agent equally cytotoxic to nonproliferating human colon adenocarcinoma cells.

Diarylsulfonylureas have been shown to have therapeutic activity against rodent and human tumor models, notably causing regressions in some lines of human colon adenocarcinomas in mice. At present the mechanism of cytotoxicity is unknown, although preliminary data implicate mitochondria as a potential site of action. In this study, the cytotoxicity of the diarylsulfonylurea N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) has been examined in GC3/c1 human colon adenocarcinoma cells. At cytotoxic concentrations of ISCU, in the presence of albumin as a drug binding species, there was only slight inhibition of [3H]thymidine and [3H]uridine incorporation at concentrations of ISCU up to 140 micrograms/ml and no inhibition of synthesis of protein as determined by incorporation of L-leucine. In the absence of albumin, incorporation of [3H]thymidine into DNA or [3H]uridine into RNA was inhibited at greater than 70 micrograms/ml and 140 micrograms/ml, respectively. As ISCU is highly bound to serum albumin (greater than 99%), it would appear that inhibition of nucleic acid synthesis occurs only at supralethal concentrations of ISCU. The cytotoxicity of ISCU in proliferating or quiescent cell populations was examined. GC3/c1 cells grown in medium containing 0.5% fetal calf serum (FCS) had a 60% reduction in rate of growth, but were more sensitive than cells exposed for 24 h in 10% FCS-medium (IC50 1.9 and 31 micrograms/ml, respectively). When the albumin concentration was adjusted (240 mg/100 ml) to allow equivalent drug binding, IC50 values were similar. In cultures of GC3/c1 cells growth rate was related to the concentration of FCS. In the absence of serum, growth rate was 2.5 to 3.2% that of exponentially growing control cultures in the presence of 10% FCS. Addition of FCS to quiescent cultures after 1 to 6 days in serum-free conditions resulted in immediate growth of cells. Clonogenic potential was also unchanged for at least 6 days under serum-free conditions. Under these conditions, where albumin concentration was adjusted to be equivalent to medium containing 10% FCS, sensitivity of proliferatively quiescent GC3/c1 cells was similar to that in exponentially growing control cultures in which the population doubling time was approximately 22 h. Further, there was no recovery of clonogenic potential when cells were exposed for 24 h to ISCU and maintained in a quiescent state for up to 4 days prior to serum stimulation. These data suggest that the cytotoxic effects of ISCU are independent of the proliferative state of the cell population.(ABSTRACT TRUNCATED AT 400 WORDS)

Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase.

Phosphatidylinositol-3-kinase is an important enzyme for intracellular signaling. The microbial product wortmannin and some of its analogues have been shown to be potent inhibitors of phosphatidylinositol-3-kinase. The 50% inhibitory concentration for inhibition by wortmannin is 2 to 4 nM. Kinetic analysis demonstrates that wortmannin is a noncompetitive, irreversible inhibitor of phosphatidylinositol-3-kinase, with inactivation being both time- and concentration-dependent. Wortmannin has previously been reported to be an inhibitor of myosin light chain kinase but with an inhibitory concentration of 0.2 microM. Wortmannin was found not to be an inhibitor of phosphatidylinositol-4-kinase, protein kinase C, or protein tyrosine kinase. Wortmannin inhibited the formation of phosphatidylinositol-3-phosphates in intact cells. The results of the study suggest that wortmannin and its analogues may have utility as pharmacological probes for studying the actions of phosphatidylinositol-3-kinase.

A novel class of monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide ribonucleotide formyltransferase and potent activity against solid tumors.

Tight-binding inhibition of recombinant human monofunctional glycinamide ribonucleotide formyltransferase by Lometrexol (6R-5,10-dideazatetrahydrofolate) requires polyglutamation. LY254155 and LY222306 differ from 5,10-dideazatetrahydrofolate in the replacement of the 1',4'- phenylene moiety by a 2',5'-thiophene and a 2',5'-furan, respectively. Compared to Lometrexol, the thiophene and furan analogues had 25- and 75-fold greater inhibitory potencies against human monofunctional glycinamides ribonucleotide formyltransferase (Ki = 2.1 and 0.77 nM, respectively). The binding affinities of the thiophene and furan analogues for membrane folate-binding protein from human KB cells were 6- and 350-fold weaker than Lometrexol, respectively. Both the thiophene analogue and 5,10-dideazatetrahydrofolate inhibited the in vivo growth of murine 6C3HED lymphosarcoma, murine C3H mammary carcinoma, and human xenograft HXGC3, HC1, and VRC5 colon carcinomas by 95-100%. The thiophene analogue was efficacious against human xenograft PANC-1, a pancreatic carcinoma which was completely resistant to 5,10- dideazatetrahydrofolate. These novel antifolates represent the first monoglutamated tight-binding inhibitors of glycinamide ribonucleotide formyltransferase. By eliminating the need for polyglutamation, this class of antifolates may have clinical activity in the treatment of solid tumors expressing low levels of folylpolyglutamate synthetase or tumors resistant to antifolate therapy due to increased gamma-glutamyl hydrolase activity.

Phase I clinical study of N-[(4-chlorophenyl)amino]carbonyl-2,3-dihydro-1H-indene-5-sulfonamide (LY186641).

Between February 1987 and July 1988, 45 patients with advanced refractory cancer were treated with LY186641, a diarylsulfonylurea that has shown a broad spectrum of activity in preclinical testing. Patients received a weekly p.o. dose of LY186641 for 6 consecutive weeks; responding and stable patients continued weekly therapy until progression occurred. Using a standard phase I study design, the first three patients received LY186641 at 30 mg/m2 week; the dose was escalated in subsequent patients until dose-limiting toxicity occurred. Methemoglobinemia was the major toxicity observed and was dose related. Methemoglobin levels peaked approximately 24 h after LY186641 was administered and fell to low levels after 48 h. Six patients developed fatigue, cyanosis, and dyspnea associated with serum methemoglobinemia levels of greater than 20%; four of these patients were subsequently removed from the study. Hemolytic anemia was also observed but was clinically significant in only 10 patients. Other side effects were mild and infrequent. The maximum tolerated dose of LY186641, when given at this schedule, was 2550 mg/m2/week. No objective tumor responses were observed.

Evidence for mitochondrial localization of N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea in human colon adenocarcinoma cells.

N-(4-Methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) is a new agent that exhibits high therapeutic activity against human and rodent tumor models. Initial studies indicated that in vitro [3H]MPCU was concentrated 4- to 6-fold in GC3/c1 human colon adenocarcinoma cells in an azide-sensitive manner. In this study the dependence of uptake and concentrative accumulation of MPCU upon temperature, plasma membrane potential, and the electrochemical potential of mitochondria has been examined. Accumulation and efflux of MPCU were temperature dependent. At 3.6 microM MPCU, initial rates of uptake (15 s) were 1.4, 38.0, and 84.2 pmol/min/10(6) cells at 2 degrees C, 23 degrees C, and 37 degrees C, respectively. The rate of uptake and concentrative accumulation within GC3/c1 cells was not altered in high K+ buffer or by 1 mM ouabain, indicating that plasma membrane potential was not significant in these processes. Concentrative accumulation, but not initial uptake, was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol, and sodium azide. Glucose partially antagonized the inhibition of these agents which uncouple oxidative phosphorylation. Oligomycin, an inhibitor of mitochondrial ATP synthase, did not inhibit uptake or concentrative accumulation of MPCU. However, oligomycin in the presence of 2-deoxyglucose significantly inhibited concentrative accumulation of MPCU. These results suggested that concentrative accumulation of MPCU was dependent upon the mitochondrial transmembrane gradient rather than ATP, although direct implication of ATP could not be excluded. To examine which component of this gradient was predominant in causing MPCU sequestration, the ionophores valinomycin and nigericin were used. Valinomycin, which collapses the charge gradient across the mitochondrial matrix membrane, caused only slight inhibition of MPCU accumulation, and the effect was similar at 2 or 10 mumol. In contrast, nigericin (which collapses the pH gradient and increases mitochondrial membrane potential) inhibited by approximately 90% concentrative accumulation of MPCU. These data suggested that MPCU was being concentrated in mitochondria and that this was dependent upon the pH gradient across mitochondrial membrane. In cells exposed to MPCU or the analogue N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, enlargement of mitochondria was observed within 24 h and appeared to be the initial morphological change associated with drug treatment. These results implicate mitochondria as a site of sequestration of diarylsulfonylureas and as a potential site of action.

Augmentation of the therapeutic activity of lometrexol -(6-R)5,10-dideazatetrahydrofolate- by oral folic acid.

Recent clinical trials with lometrexol [(6R)-5,10-dideazatetrahydrofolate] have revealed a level of toxicity in humans that was not predicted on the basis of previous in vivo preclinical studies. Because standard laboratory animal diets contain high levels of folic acid relative to human folate intake, the toxicity and therapeutic activity of lometrexol was studied in mice under conditions of restricted dietary folate intake. Remarkably, the lethality of this drug increased by three orders of magnitude in mildly folate-deficient mice, mimicking the unexpected toxicity seen in humans. Lometrexol had limited therapeutic activity in folate-deficient mice bearing the C3H mammary adenocarcinoma, compared with the substantial therapeutic index for treatment of this tumor in animals on standard diet. When folic acid was administered p.o. to mice that were mildly folate deficient, antitumor activity was again observed at nontoxic doses of lometrexol, and the range of lometrexol doses that allowed safe therapeutic use of this drug increased at higher dietary folate intake. At a fixed dose of lometrexol, the antitumor effects in animals were dependent on the level of dietary folate and went through a distinct optimum. Excessively high folate intake reversed the antitumor effects of lometrexol. Optimization of the folic acid content in the diet and of the lometrexol dosage are predicted to have substantial impact on the clinical activity of this class of drugs.

Phase I study of high-dose methotrexate with thymidine and low-dose leucovorin.

A total of 15 patients with advanced neoplastic disease, 13 with different solid tumors, one with lymphoma, and one with acute lymphocytic leukemia, underwent treatment consisting of continuous infusion of methotrexate (2 g/sq m/day) with concomitant thymidine (8 g/sq m/day) and leucovorin (1 mg/sq m/day). The dose of methotrexate was increased progressively by lengthening the methotrexate infusion from 2 to 7 days. After cessation of methotrexate infusion, thymidine and leucovorin were continued until the plasma level of methotrexate decreased to 2 X 10(-8) M. Toxicity was mucositis (23 of 27 evaluable courses), leukopenia (15 of 26 evaluable courses), thrombocytopenia (10 of 26 evaluable courses), renal and hepatic toxicity and diarrhea. Plateau levels of plasma methotrexate or methotrexate plasma half-life did not correlate with toxicity.

Characterization of folate receptor from normal and neoplastic murine tissue: influence of dietary folate on folate receptor expression.

Membrane-associated folate receptors (FRs) have been detected in many mammalian species, and multiple isoforms have been identified. The pharmacological properties of FRs from murine kidney, liver, and six murine tumors were characterized. Murine kidney expressed primarily folate-binding protein 1, analogous to human FR-alpha, whereas murine liver expressed predominantly folate-binding protein 2, analogous to human FR-beta. Five of six murine tumors expressed high-affinity FRs with pharmacological properties consistent with folate-binding protein 1 isoform expression. Restriction of dietary folate resulted in significant changes in the FR expression in most murine tissues. Kidney and tumor FRs showed a decreased affinity for folic acid, suggesting a change in isoform expression in response to a low folate diet. Density of the FR in the kidney decreased, and, in contrast, density of the FR in all tumors increased. The response of the liver to a low folate diet was unique in that there were no detectable changes in affinity or density of liver FR. Changes in dietary folate that modulate FR isoform expression may have relevance for cancer patients treated with antifolates.