Flunarizine enhancement of melphalan activity against drug-sensitive/resistant rhabdomyosarcoma.

Flunarizine, a diphenylpiperazine calcium channel blocker, is known to increase tumor blood flow. It also interferes with calmodulin function, repair of DNA damage and drug resistance associated with P-glycoprotein. Flunarizine was tested for its ability to modulate either cyclophosphamide- or melphalan-induced growth delay for a drug-resistant rhabdomyosarcoma xenograft (TE-671 MR) and the drug-sensitive parent line (TE-671), in which P-glycoprotein is not involved in the mechanism of drug resistance. Tumour blood flow was increased by 30% after a flunarizine dose of 4 mg kg-1, but no modification in growth delay was induced by melphalan (12 mg kg-1). In contrast, a 60 mg kg-1 dose of flunarizine had no effect on tumour blood flow, but the same dose created significant enhancement in melphalan-induced tumour regrowth delay in both tumour lines. The dose-modifying factor for flunarizine as an adjuvant to melphalan was approximately 2 for both tumour lines. Although blood flow measurements were not performed with the combination of flunarizine and melphalan, the results from flunarizine alone suggested that augmentation of melphalan cytotoxicity is not mediated by changes in blood flow. In contrast, flunarizine did not affect drug sensitivity to cyclophosphamide in groups of animals bearing the drug-sensitive parent tumour line. These results suggest that the mechanism of drug sensitivity modification by flunarizine is not related to modification of tumour blood flow, but may be mediated by modification of transport mechanisms that are differentially responsible for cellular uptake and retention of melphalan as compared with cyclophosphamide.

Effects of glutathione or polyamine depletion on in vivo thermosensitization.

Investigations with the melphalan-sensitive and -resistant human rhabdomyosarcoma xenografts TE-671 and TE-671 MR were performed to examine the effect of glutathione and polyamine modulation on thermosensitivity. Regimens of intraperitoneally injected and orally administered buthionine sulfoximine were utilized to achieve glutathione depletion to 8.7% and 13% of control levels in TE-671 and TE-671 MR, respectively. Animals treated with L-buthionine-S,R-sulfoximine and 42 degrees C or 43 degrees C hyperthermia for 70 min showed no detectable growth delays beyond those observed for hyperthermia alone. Hyperthermia at 42 degrees C of disaggregated TE-671 and TE-671 MR xenografts following growth in short-term culture was performed following preincubation with buthionine sulfoximine or 0.9% saline. Buthionine sulfoximine-mediated glutathione depletion produced a significant increase in hyperthermia-induced cytotoxicity only with TE-671 MR at 43 degrees C. Polyamine depletion was achieved with a 7-day orally administered course of MDL 72.175DA [(2R,5R)-6-heptyne,5-diamine dihydrochloride], an irreversible inhibitor of ornithine decarboxylase. Although this treatment caused significant depletion of intracellular putrescine and spermidine levels, spermine levels remained relatively unaffected. No significant growth delays were observed in either xenograft line for animals treated with MDL 72.175DA or MDL 72.175DA plus hyperthermia as compared with untreated controls. These results contrast with previous work performed in vitro showing synergism between glutathione or polyamine depletion and hyperthermia, and indicate that further studies are needed.

Hyperthermia-induced enhancement of melphalan activity against a melphalan-resistant human rhabdomyosarcoma xenograft.

The effects of regional hyperthermia (42 degrees C for 70 min) on the antitumor activity of melphalan were examined in athymic mice bearing melphalan-resistant human rhabdomyosarcoma (TE-671 MR) xenografts growing in the right hind limb, and results were compared with similar studies of melphalan-sensitive (TE-671) parent xenografts. Melphalan alone at a dose of 36 mg/m2 (0.5 of the 10% lethal dose) produced growth delays of 4.1 to 10.2 days in TE-671 MR xenografts and 21.8 to 28.7 days in TE-671, respectively. Hyperthermia alone produced growth delays of 0.9 days in TE-671 MR xenografts and 0.8 days in TE-671. Combination therapy with melphalan and hyperthermia produced growth delays of 7.2 to 13.3 days in TE-671 MR xenografts and 34.3 to 42.8 days in TE-671, respectively, representing a mean thermal enhancement ratio of 1.7 in TE-671 MR and 1.5 in TE-671. Measurement of glutathione levels in TE-671 MR xenografts following treatment with melphalan, hyperthermia, or melphalan plus hyperthermia revealed significant reductions in glutathione content with the nadir (60% of control values) seen 6 h following treatment. Glutathione levels in TE-671 xenografts following identical therapy revealed no differences from control values. Hyperthermia plus melphalan did not result in a higher tumor-to-plasma melphalan ratio compared with treatment with melphalan alone in either TE-671 MR or TE-671 xenografts. These studies suggest that heat-induced alterations in tumor glutathione or melphalan levels are not responsible for the increase in melphalan activity produced by hyperthermia. Combination therapy with melphalan plus regional hyperthermia offers promise for treatment of melphalan-resistant neoplasms.

Enhancement of melphalan-induced gastrointestinal toxicity in mice treated with regional hyperthermia and BSO-mediated glutathione depletion.

Both hyperthermia and glutathione depletion have been shown to increase the antineoplastic activity of melphalan. Investigations were carried out to define the toxicity and activity of melphalan given in conjunction with local (right hind limb) hyperthermia and L-buthionine-SR-sulphoximine (BSO)-mediated glutathione depletion to athymic mice bearing the melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR. Administration of 0.5 of the 10% lethal dose of melphalan to mice treated with BSO and hyperthermia (42 degrees C for 70 min) resulted in a 53% mortality rate. The mortality rates for mice treated with melphalan alone (2.5%), hyperthermia alone (0%), melphalan plus BSO (13.5%), melphalan plus hyperthermia (12.0%) and BSO plus hyperthermia (0%) were substantially lower than triple therapy. Histological examination of kidney, liver, colon, and small intestine sections taken from non-tumour-bearing animals revealed a marked increase in damage to the small intestine (cryptal necrosis and epithelial denudement) in animals receiving triple therapy compared with animals receiving any other treatment combination. Gavage administration of sterile water (1 ml twice a day) completely prevented mortality in animals receiving triple therapy. Treatment of tumour-bearing animals with triple therapy plus gavage demonstrated a statistically significant increase in tumour growth delay compared with animals receiving any other treatment combination.

Influence of glutamine on the growth of human glioma and medulloblastoma in culture.

Cellular supply of glutamine, an essential substrate for growth, is derived from extracellular fluid and de novo synthesis. We investigated the relative importance of these sources to the growth of six human anaplastic glioma- and one human medulloblastoma-derived permanent cell lines. Exogenous glutamine was limiting for the proliferation of glioma-derived lines D-54 MG, U-118 MG, and U-251 MG. In contrast, medulloblastoma-derived line TE-671 and glioma-derived lines U-373 MG, D-245 MG, and D-259 MG grew in the absence of supplemental glutamine. Two cell lines with contrasting glutamine requirements, D-54 MG and TE-671, were used to explore the pharmacological interference with glutamine metabolism. DL-alpha-Aminoadipic acid, a reported glutamic acid analogue with gliotoxic properties, significantly inhibited the growth of both lines. These effects were reversed by increasing glutamine, suggesting that the major action of DL-alpha-aminoadipic acid is as a glutamine antagonist. In contrast, the glutamine synthetase inhibitor delta-hydroxylysine demonstrated activity only against TE-671. Acivicin and 6-diazo-5-oxo-L-norleucine, glutamine analogues available for clinical use, reduced the proliferation of both cell lines at pharmacological concentrations. Methionine sulfoximine, a glutamine synthetase inhibitor previously used clinically, produced marked growth inhibition only against TE-671. These findings indicate that the synthesis and utilization of glutamine are potentially exploitable targets for the chemotherapy of some human gliomas and medulloblastomas.

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 3. Synthesis and activity of some nucleosides of 4-[(arylalkenyl)thio]pyrazolo[3,4-d]pyrimidines.

Ribonucleosides of 4-(alkylthio)-1H-pyrazolo[3,4-d]pyrimidines have been shown to be useful anticoccidial agents [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32. Rideout, J. L.; Krenitsky, T. A.; Elion, G. B. U.S. Patent 4299 283, 1981]. In that study, the unsaturated 4-allylthio and 4-crotylthio derivatives (19 and 20) were shown to be more active in vivo against Eimeria tenella than their saturated congeners; therefore, some unsaturated (arylalkyl)thio derivatives were synthesized and investigated as anticoccidial agents. The novel compounds in this study (2 to 18) were prepared by the alkylation of 4-mercapto-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (1), which was prepared by an enzymatic method. The (E)-4-cinnamylthio derivative (2) and the 5'-monophosphate (18) were the most active compounds against E. tenella in vivo. None of the analogues with substituents in the aryl moiety (3 to 13) was more active than 2 in vivo. The geometry about the double bond was important, since the (Z)-4-cinnamylthio derivative (14) was inactive both in vitro and in vivo. The 4-(3-phenylpropynyl)thio and 4-(5-phenyl-2,4-pentadienyl)thio derivatives (15 and 16) were at least as active as 2 in vitro; however, they were less active than 2 in vivo. Compound 2 was effective in vivo against E. tenella, E. necatrix, E. maxima, and E. brunetti; these species of Eimeria were controlled when 2 was given in the diet at levels upt to 100 ppm. Infections in vivo due to E. acervulina were controlled by 2 only at about 800 ppm. The broad spectrum of anticoccidial activity shown by 2 represents a significant improvement over the activities reported for related compounds [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32].

Modulation of cyclic AMP metabolism by S-adenosylhomocysteine and S-3-deazaadenosylhomocysteine in mouse lymphocytes.

Mouse lymphocytes incubated with micromolar concentrations of adenosine or 3-deazaadenosine, in medium supplemented with L-homocysteine, rapidly accumulated supramillimolar concentrations of S-adenosylhomocysteine (AdoHcy) or S-3-deazaadenosylhomocysteine (c3AdoHcy), respectively. Lymphocytes thus preloaded with high levels of AdoHcy or c3AdoHcy exhibited markedly enhanced (5- to 40-fold) cyclic AMP responses to prostaglandin E1, adenosine, 2-chloroadenosine, isoproterenol, and cholera toxin. This enhancement of cyclic AMP response by intracellular AdoHcy or c3AdoHcy was attributable both to amplification of the activity of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and to inhibition of cyclic AMP phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17). Basal and prostaglandin E1- and isoproterenol-stimulated activities of adenylate cyclase, assayed in lymphocyte homogenates, were increased 1.3- to 2.0-fold after treatment of the cells with homocysteine plus either adenosine or 3-deazaadenosine. AdoHcy and c3AdoHcy were found to be competitive inhibitors (with Ki values of 1.7 and 4.8 mM, respectively) of the high-affinity cyclic AMP phosphodiesterase present in lymphocyte homogenates. It is evident, therefore, that increased cellular levels of AdoHcy or c3AdoHcy can affect cellular physiology via perturbation of cyclic AMP metabolism as well as via inhibition of S-adenosylmethionine-dependent methylation reactions.