Cell cycle modulation by a multitargeted antifolate, LY231514, increases the cytotoxicity and antitumor activity of gemcitabine in HT29 colon carcinoma.

The proliferation rate of HT29 colon carcinoma cells was decreased by the multitargeted antifolate (MTA), LY231514. This effect correlated with a buildup of cells near the G1-S interface after 24 h of incubation, and a synchronized progression of the population through S phase during the next 24 h. MTA treatment (0.03-3 microM) was minimally cytotoxic (20-30%) to HT29 cells after a 24-h exposure, and no dose response was observed. In contrast, the nucleoside analogue gemcitabine (GEM) was cytotoxic (IC50, 0.071 +/- 0.011 microM; IC90, 0.648 +/- 0.229 microM) after a 24-h exposure. We hypothesized that pretreatment of these cells with MTA would increase the potency of GEM by synchronizing the population for DNA synthesis. The cytotoxicity of GEM increased 2-7-fold when MTA was administered 24 h before GEM (IC50, 0.032 +/- 0.009 microM; IC90, 0.094 +/- 0.019 microM). In addition, an increase in cell kill for the combination compared with GEM alone (IC99, 12 microM for GEM alone; IC99, 0.331 microM for combination) was observed. No increase in potency or cell kill was observed when the two compounds were added simultaneously. MTA pretreatment also potentiated the cytotoxicity of a 1-h exposure to GEM. These cell-based observations were extended to evaluate the schedule-dependent interaction of these two agents in vivo using a nude mouse HT29 xenograft tumor model. At the doses tested, MTA alone (100 mg/kg) had a marginal effect on tumor growth delay, whereas GEM (80 mg/kg) produced a statistically significant tumor growth delay. In combination, the increase in tumor growth delay was greatest when MTA was administered before GEM, compared with simultaneous drug administration or the reverse sequence, e.g., GEM followed by MTA. The effect of sequential administration of MTA followed by GEM was greater than additive, indicating synergistic interaction of these agents. Thus, in vitro, MTA induced cell cycle effects on HT29 cells that resulted in potentiation of the cytotoxicity of GEM. In vivo, combination of these two drugs also demonstrated a schedule-dependent synergy that was optimal when MTA treatment preceded GEM.

Role of thymidylate synthase in the antitumor activity of the multitargeted antifolate, LY231514.

The cytotoxicity of LY231514 was only partially alleviated by thymidine addition (5 microM) in GC3 human colon carcinoma cells, and complete protection required the addition of both hypoxanthine (100 microM) and thymidine. In contrast, the cytotoxic activity of tomudex (raltitrexed, ZD1694) was completely reversed by thymidine alone. MCF-7 human breast and H630 human colon carcinoma cells selected for resistance to tomudex and 5-fluorouracil, respectively via thymidylate synthase (TS) amplification demonstrated only modest resistance to LY231514 compared to tomudex. LY231514-induced cytotoxicity in these resistant cell lines was completely prevented by the addition of hypoxanthine (100 microM), indicating inhibition of purine de novo biosynthesis as a secondary target for LY231514 action. Thymidine at physiologic levels in mouse plasma (approximately 1 microM) produced only a 2.6-fold shift in the IC50 for LY231514-mediated cytotoxicity in GC3/cl1 cells compared to a 128-fold shift for tomudex. LY231514 treatment (i.p., qd x 10) significantly delayed tumor growth in the GC3 carcinoma xenograft model. However, a thymidine kinase-deficient mutant of this same tumor line demonstrated heightened sensitivity to the in vivo antitumor activity of LY231514 with complete regression of established tumors and a large number of tumor-free survivors after one course of treatment. The data demonstrate that inhibition of thymidylate synthase is a prominent mechanism for antitumor activity by LY231514, but important secondary sites of action exist for this multitargeted molecule.

The synthesis and biological activity of a series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates as antineoplastic and antiarthritic agents.

A new series of 2,4-diaminopyrido[2,3-d]pyrimidine based antifolates 1-3 were synthesized through an efficient conversion of 2-pivaloyl-4-oxo-6-ethynylpyrido[2,3-d]pyrimidine 5 to the corresponding 4-amino analog 7 via the activated 1,2,4-triazole intermediate 6. Compound 7 was used as the key intermediate for the preparation of the final products. The detailed biological evaluation of these compounds both as antineoplastic and antiarthritic agents will be discussed.

Synthesis and biological evaluation of novel cryptophycin analogs with modification in the beta-alanine region.

Structure modification of the beta-alanine region (fragment C) of the potent antimitotic agent cryptophycin was investigated. This includes: (1) introduction of substituents at the previously unsubstituted C7 position of the macrolide ring and (2) replacement of the (2R)-3-amino-2-methyl-propanoic acid (beta-alanine) with various (1)-amino acids to give the corresponding 15-membered unnatural cryptophycin analogs.

Role of folic acid in modulating the toxicity and efficacy of the multitargeted antifolate, LY231514.

We studied the effects of folic acid on modulating the toxicity and antitumor efficacy of LY231514. Using several human tumor cell lines adapted to growth in low folate medium, folic acid was shown to be 100- to 1000-fold less active than folinic acid at protecting cells from LY231514-induced cytotoxicity. The lethality of LY231514 was compared in mice maintained on standard diet or low folate diet. The LD50 occurred at 60- and 250-fold lower doses of LY231514 in DBA/2 and CD1 nu/nu mice, respectively, maintained on low folate diet compared to standard diet. The L5178Y/TK-/HX-murine lymphoma was much more sensitive to the antitumor action of LY231514 compared to wild type L5178Y-S tumors. For mice on low folate diet, LY231514 at 0.3 and 1 mg/kg (qd x 10, i.p.) produced 100% inhibition of L5178Y/TK-/HX-lymphoma growth, and significant lethality occurred at > or = 3 mg/kg. For mice on standard diet, LY231514 produced > 95% inhibition of tumor growth at 30 to 300 mg/kg, but all mice died at 800 mg/kg. Folic acid supplementation was demonstrated to preserve the antitumor activity of LY231514 while reducing toxicity. The combination of folic acid with LY231514 may provide a mechanism for enhanced clinical antitumor selectivity.

Whole-body disposition and polyglutamate distribution of the GAR formyltransferase inhibitors LY309887 and lometrexol in mice: effect of low-folate diet.

PURPOSE: The whole-body autoradiographic distribution of two radiolabeled antifolate inhibitors of GAR formyltransferase, lometrexol and LY309887, were compared in tumor-bearing mice maintained on standard diet (SD) and a low-folate diet (LFD) in order to determine the total amounts of drug that accumulated in blood, tumor, liver and kidney. The time-dependent changes in tissue distribution were evaluated over a 7-day period in order to compare the pharmacokinetic properties of both inhibitors and to assess the influence of dietary folate on this distribution. In addition, the effect of dietary folate on polyglutamation of compound accumulating in the liver was measured. The results have bearing on the potential of these two clinical agents to produce delayed toxicity in cancer patients and the use of dietary folate to modulate or prevent the development of this toxicity. METHODS: Single equimolar i.v. doses of [14C]LY309887 and [14C]lometrexol were administered to C3H mammary tumor bearing mice on SD or LFD, and the disposition of these compounds was quantitated using whole-body autoradiography. Livers were also harvested and extracted for determination of polyglutamate distribution. Animals were sacrificed both early and late (7 days) after dosing to determine the long-term retention of these compounds. RESULTS: Whole-body autoradiography revealed that the highest concentrations of both compounds were in liver and kidney. Concentrations of both compounds were two-fold higher in livers from LFD mice than in livers from SD mice. Lometrexol concentrations in liver averaged 2.8- and 2.2-fold higher than LY309887 in SD and LFD livers, respectively. In SD livers, the polyglutamate profiles of both compounds were similar, with hexaglutamates being the longest chain species detected. In LFD livers, hexaglutamates of LY309887 were observed, while hepta- and octaglutamates of lometrexol were detected after 168 h. CONCLUSIONS: The reduced hepatic retention and biochemical profile of LY309887 compared to lometrexol suggest that it may be less likely to produce delayed cumulative toxicity while still retaining antitumor activity. However, the increased hepatic accumulation observed in LFD mice emphasizes the importance of assessing and supplementing folate in cancer patients treated with this class of compounds.

Sulfonimidamide analogs of oncolytic sulfonylureas.

A series of sulfonimidamide analogs of the oncolytic diarylsulfonylureas was synthesized and evaluated for (1) in vitro cytotoxicity against CEM cells, (2) in vivo antitumor activity against subaxillary implanted 6C3HED lymphosarcoma, and (3) metabolic breakdown to the o-sulfate of p-chloroaniline. The separated enantiomers of one sulfonimidamide analog displayed very different activities in the in vivo screening model. In general, several analogs demonstrated excellent growth inhibitory activity in the 6C3HED model when dosed orally or intraperitoneally. A correlative structure-activity relationship to the oncolytic sulfonylureas was not apparent.

Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts.

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr.mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/hr.mg protein), liver (109 pmol/hr.mg protein), murine C3H mammary tumor (112 pmol/hr.mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr.mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25-50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

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.

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.

Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs.

Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC50 = 4.2 nM) of phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several intracellular signaling pathways, structure-activities studies on wortmannin analogs were performed in an effort to understand the structural requirements necessary for PI 3-kinase inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase inhibition. We have prepared various wortmannin analogs which address the possibility of this mechanism. Of particular interest are compounds which affect the C-21 position of wortaminnin either sterically or electronically. Our results support the conclusion that nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring analogs of wortmannin, and their activities are reported herein. We conclude that the wortmannin D ring is an important recognition site since modifications have such a dramatic effect on inhibitor potency. Finally, the identification of 17beta-hydroxywortmannin represents the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.

Biochemistry and pharmacology of glycinamide ribonucleotide formyltransferase inhibitors: LY309887 and lometrexol.

Lometrexol, a tight-binding antifolate inhibitor of the purine de novo enzyme glycinamide ribonucleotide formyltransferase (GARFT), was the first GARFT inhibitor to be investigated clinically. Unexpected observations of delayed cumulative toxicity prompted a search for a second generation antimetabolite with a more favorable biochemical, pharmacological and toxicological profile. LY309887, 6R-2',5'-thienyl-5, 10-dideazatetrahydrofolic acid, had 9-fold greater potency to inhibit GARFT (Ki = 6.5 nM) compared to lometrexol. Like lometrexol, LY309887 was activated by folpolyglutamate synthetase, however, it had a lower first order rate constant. In vitro and in vivo data were consistent with these observations: polyglutamation of LY309887 was less extensive compared to lometrexol and livers of mice accumulated fewer polyglutamates of LY309887 than polyglutamates of lometrexol. The affinities of these two compounds for isoforms of human folate receptors (FR) were compared. Lometrexol had a 6-fold higher affinity for FR alpha than LY309887 and both compounds had higher affinity for the alpha isoform compared to the beta isoform. The selectivity of LY309887 for FR alpha (beta (Ki)/ alpha (Ki) = 10.5) was twice that of lometrexol's (beta / alpha = 5.0). Lometrexol and LY309887 were potent cytotoxic compounds against the human leukemia cell line CCRF-CEM with IC50's of 2.9 nM and 9.9 nM, respectively. In vivo, LY309887 was more potent than lometrexol at inhibiting tumor growth in the C3H mammary murine tumor model and several tumor xenografts. Excellent efficacy was achieved by both compounds in several colon xenografts. In two pancreatic human xenografts, LY309887 achieved greater efficacy than lometrexol. In summary, the biochemical and pharmacological properties of lometrexol and LY309887 support the hypothesis that these antifolates will have clinical activity against human solid tumors. LY309887 is a second generation GARFT inhibitor with biochemical and pharmacological properties which distinguish it from lometrexol and suggest that it will have broad antitumor activity, a different pharmacokinetic profile and produce less toxicity than lometrexol in cancer patients.

Lack of in vivo crossresistance with gemcitabine against drug-resistant murine P388 leukemias.

Gemcitabine, a novel pyrimidine nucleoside antimetabolite, has shown clinical antitumor activity against several tumors (breast, small-cell and non-small-cell lung, bladder, pancreatic, and ovarian). We have developed a drug-resistance profile for gemcitabine using eight drug-resistant P388 leukemias in order to identify potentially useful guides for patient selection for further clinical trials of gemcitabine and possible noncrossresistant drug combinations with gemcitabine. Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin or etoposide) exhibited no crossresistance to gemcitabine. Leukemias resistant to vincristine (not multidrug resistant), cyclophosphamide, melphalan, cisplatin, and methotrexate were also not crossresistant to gemcitabine. Only the leukemia resistant to 1-beta-D-arabinofuranosylcytosine was crossresistant to gemcitabine. The results suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-beta-D-arabinofuranosylcytosine and (2) the lack of crossresistance seen with gemcitabine may contribute to therapeutic synergism when gemcitabine is combined with other agents.

Pharmacokinetics of the anticancer agent sulofenur in mice, rats, monkeys, and dogs.

The absorption and pharmacokinetics of sulofenur [N-(indan-5-sulfonyl)-N'-(4-chlorophenyl)urea, LY186641] and its major metabolites were examined in mice, rats, monkeys, and dogs. The compound is a diarylsulfonylurea currently being evaluated as an oncolytic agent in phase I and II trials. In all species, sulofenur was well absorbed after an oral dose, but over a prolonged period, and sulofenur exhibited a fairly long half-life of elimination from plasma. These values ranged from 6 h in rats up to 30, 110, and 200 h in mice, monkeys, and dogs, respectively, at doses (240-1000 mg/m2) within the range of those used in clinical trials. Experiments describing the high degree of binding of sulofenur to plasma proteins (consistently > 99%) help to explain these relatively long half-lives. There is, however, a large difference between these plasma half-lives in the species studied. Sulofenur was previously found to be extensively metabolized to products that are excreted primarily into the urine. In this study, its major metabolites, which are found mainly in the urine, were also minor components of the drug-related material (< 10% of the sulofenur concentrations) in the plasma of rats treated with sulofenur. The absorption, binding characteristics, and elimination of these major metabolites after their administration to rats were also compared with sulofenur.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of metabolism and toxicity to the structure of the anticancer agent sulofenur and related sulfonylureas.

The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.

Synthesis and biological activity of acyclic analogues of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis and biological evaluation of a number of analogues of N-[4-[4-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidyl) butyl]benzoyl]-L-glutamic acid (2) (7-DM-DDATHF), an acyclic modification of the novel folate antimetabolite 5,10-dideazatetrahydrofolic acid (DDATHF), are described. The synthetic procedure utilized previously for the synthesis of 2, 15, and 16 was extended to the preparation of analogues modified in the benzoyl region with thiophene and methylene groups replacing the benzene ring (compounds 27a-c) and in the glutamate region with aspartic acid and phenylalanine replacing L-glutamic acid (compounds 36, 37). The 2-amino-4,6-dioxo derivative 33 was obtained from intermediate 30 via a palladium-catalyzed carbon-carbon coupling reaction with diethyl (4-iodobenzoyl)-L-glutamate, followed by reduction and removal of protecting groups with base. Cell culture cytotoxicity studies of all of the above acyclic analogues of DDATHF against CCRF-CEM human lymphoblastic leukemic cells gave IC50s ranging from 0.042 greater than 48 microM. Inhibition and cell culture reversal studies against isolated enzymes suggest the mode of action of these compounds. Compound 2 was only 3-fold less inhibitory toward glycinamide ribonucleotide formyltransferase (GARFT, isolated from L1210 leukemic cells) than DDATHF itself. These acyclic analogues were less efficient substrates for the enzyme folylpolyglutamate synthetase (FPGS) compared with their bicyclic counterparts. Moderate antitumor activity was observed for compound 2 against 6C3HED lymphosarcoma and C3H mammary adenocarcinoma in vivo.

Automated measurement of transplantable solid tumors using digital electronic calipers interfaced to a microcomputer.

Data collection for transplantable solid tumors has been automated with electronic digital calipers and a balance which have been coupled through an RS-232 interface to a microcomputer. BASIC programs handle data entry, calculations and data storage. A "PROTOCOL" program accepts keyboard input of sample name, notebook number, submitter and dose along with necessary information on tumor system, and then initial animal weights for treatment groups are sent from balance to computer. Data is stored as an ASCII file on floppy disks, and protocol reports are printed. When the test is to be measured, a "MEASURE" program prompts the user for keyboard entry of toxic deaths in each group. Then the computer requests input of width and length of tumors for each animal. These tumor dimensions are sent to microcomputer by pressing a button on the calipers. When a group is completed, final animal weights are sent from balance to microcomputer. Then tumor weights and percent inhibition as compared to appropriate control groups are calculated, and the data is appended to the file for that test. A hard copy is generated as tumors are measured, and reports including percent inhibition can be printed immediately after a test is measured. The data as an ASCII file is transferred via modem to mainframe computer, where another program transfers the information to a database management program. These automated procedures for tumor measurement save time and lessen the chance for error by eliminating manual recording of solid tumor dimensions and subsequent reentry of this data for calculation.