John F. Worzalla

The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Preclinical and Clinical Evaluation of the Glycinamide Ribonucleotide Formyltransferase Inhibitors Lometrexol and LY309887

The importance of the purine de novo pathway in providing DNA precursors for cancer cell growth led to the hypothesis that novel antifolate inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), the first folate-dependent enzyme in this pathway, might have utility in the treatment of cancer. In 1987, clinical investigations were initiated with lometrexol (6R-dideazatetrahydrofolic acid, 6R-DDATHF), a novel “tight-binding” inhibitor of GARFT with potent antitumor activity in a number of murine and human xenograft solid tumors. Unexpected observations of delayed cumulative toxicity in phase I clinical trials prompted extensive preclinical investigations of the dynamics of folate status on the efficacy and toxicity of GARFT inhibitors and other antifolates (1). In addition, structure-activity studies have led to the identification of a second generation GARFT inhibitor, LY309887 (2´, 5´-thienyl-dideazatetrahydrofolic acid), which is more potent than lometrexol and has greater antitumor efficacy in vivo (2). Biochemical and pharmacological differences between LY309887 and lometrexol with respect to potency to inhibit GARFT, differential transport and storage in liver, and polyglutamation suggest that LY309887 may have greater antitumor efficacy and more manageable toxicity in the clinic than lometrexol. A murine model of the delayed cumulative toxicity seen with lometrexol has been refined and characterized to provide greater understanding of the pharmacokinetics and pharmacodynamics of these events. In concert with recently published nutritional data on the folate status of humans and more sophisticated methods of assessing and modulating antifolate toxicities through vitamin supplementation, antifolate therapy may be poised to enter a new phase of clinical success. In this report, we describe LY309887, a GARFT inhibitor with unique biochemical and pharmacological properties that has antitumor activity against a broad panel of human xenograft tumors, and greater potency than lometrexol both as an inhibitor of GARFT and as an inhibitor of tumor growth in vivo. An overview of the phase I clinical results with lometrexol and the design of the phase I clinical trial with LY309887 will be presented.

Plasma levels and urinary excretion of hexamethylmelamine following oral administration to human subjects with cancer

An ultraviolet spectrophotometric analytical method was used to study the plasma levels and urinary metabolites of cancer patients following oral administration of the antineoplastic agent hexamethylmelamine (HMM). Maximum plasma levels varied directly with the dose administered and occurred 2 to 3 hours after drug ingestion. At clinically employed dosage levels no intact HMM was present in the plasma, but two metabolites of this drug were detected. Hemolyzed erythrocytes contained no HMM or drug metabolites. Nearly 19 per cent of a single oral dose of HMM was excreted in the urine 0 to 24 hours after administration, with an additional 12 per cent appearing between 24 and 48 hours. Following prolonged continual daily administration of HMM, 50 to 65 per cent of a daily dose was recovered from the urine as drug metabolites. At least 3 maior urinary metabolites of HMM were found in urine, but no unmetabolized HMM was recovered. These data suggest that the metabolism of HMM may be significantly different from that of its structural analogue triethylenemelamine.

Synthesis and biological evaluation of a new series of dihydrofolate reductase inhibitors based on the 4-(2,6-diamino-5-pyrimidinyl)alkyl-L-glutamic acid structure

Abstract A novel series of dihydrofolate reductase inhibitors was uncovered during an expansion of the SAR of 5,10-dideazatetrahydrofolic acid, and their biological activity was evaluated. These new analogs do not possess an oxygen at the 4 position and contain a monocyclic pyrimidine ring.

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.

Inhibition of intracellular Ca2+ signalling, cytotoxicity and antitumor activity of the herbicide oryzalin and its analogues

Purpose: Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca2+ ([Ca2+]i) signalling in mammalian cells. Methods and Results: Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca2+]i signalling in Swiss 3T3 fibroblasts with IC50 values of 14, 16 and 18 μM, respectively. 45Ca2+ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC50 of 34 μM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC50 of 8 μM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC50 values in the range 3 to 22 μM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca2+]i signalling and inhibition of the growth of HT-29 human colon cancer cells (P=0.001) and of CCRF-CEM human leukemia cells (P=0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 μM and of its major metabolite N-depropyl oryzalin was 53 μM. Conclusion: It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations.

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.