Serotonin involvement in the antitumour and host effects of flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

The relationship of serotonin (5-HT) receptors to the action of the experimental antitumour drugs flavone-8-acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) was studied. Both FAA and 5,6-MeXAA are known to induce the synthesis of tumour necrosis factor- (TNF) and to stimulate nitric oxide synthesis in vivo, as measured by elevation of plasma nitrate. Serotonin potentiated the effect of a subtherapeutic dose of 5,6-MeXAA (20 mg/kg) as measured both by plasma nitrate increase and by growth delay of s.c. implanted colon 38 tumours. On the other hand, administration of the serotonin 5-hydroxytryptamine-2 (T-HT₂) antagonist cyproheptadine (20 mg/kg) inhibited both the plasma nitrate response and, to a lesser extent, the induction of tumour haemorrhagic necrosis by 5,6-MeXAA, FAA and TNF. Reduction of circulating plasma serotonin by pre-treatment withp-chlorophenylalanine and reserpine reduced the plasma nitrate response, but not the tumour necrosis response, to 5,6-MeXAA (30 mg/kg). It is suggested that serotonin is necessary for the induction of nitric oxide synthases and acts, either directly or indirectly, in concert with TNF. Serotonin agonists may have utility in increasing nitric oxide synthesis in response to TNF or to agents that induced TNF as part of their antitumour action.This research was supported by the Auckland Division of Cancer Society of New Zealand and the Health Research Council of New Zealand, by the Health Research Council of New Zealand and by the Ruth Spencer Medical Research Fellowship Trust.     

Plasma pharmacokinetics of the antitumour agents 5,6-dimethylxanthenone-4-acetic acid,xanthenone-4-acetic acid and flavone-8-acetic acid in mice

Summary Although the antitumour agent flavone-8-acetic acid (FAA) exhibits remarkable activity against murine solid tumours, its clinical use has a number of pharmacological drawbacks, including low dose potency and dose-dependent pharmacokinetics. Xanthenone-4-acetic acid (XAA) and its 5,6-dimethyl derivative (5,6-MeXAA) were synthesised during a search for better analogues of FAA. The maximal tolerated doses (MTDs) of 5,6-MeXAA, XAA and FAA in BDF₁ mice were 99, 1,090 and 1,300 mol/kg, respectively. At the MTD, 5,6-MeXAA displayed the following pharmacokinetic properties: maximal plasma concentration, 600 M; mean residence time, 4.9 h; AUC, 2,400 mol h l^–1; and volume of steady-state distribution, 0.2 l/kg. All compounds displayed nonlinear elimination kinetics at the MTD, but when the logarithm of the AUC was plotted against that of the delivered dose, the slope of the regression line for 5,6-MeXAA was found to be 1.2 as opposed to 1.4 for XAA and 1.98 for FAA. 5,6-MeXAA thus showed only a slight deviation from dose-independent kinetics. 5,6-MeXAA bound to plasma proteins in a manner similar to that exhibited by FAA, although the plasma concentration of free drug was lower for the former than for the latter. As a consequence, the calculated maximal free drug concentration for 5,6-MeXAA in plasma was 23 times lower than that for FAA.This work was supported by a Todd Foundation Clinical Oncology Research Training Fellowship, by the Medical Research Council of New Zealand and by the Cancer Society of New Zealand     

Serotonin involvement in the antitumour and host effects of flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

The relationship of serotonin (5-HT) receptors to the action of the experimental antitumour drugs flavone-8-acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) was studied. Both FAA and 5,6-MeXAA are known to induce the synthesis of tumour necrosis factor-α (TNF) and to stimulate nitric oxide synthesis in vivo, as measured by elevation of plasma nitrate. Serotonin potentiated the effect of a subtherapeutic dose of 5,6-MeXAA (20 mg/kg) as measured both by plasma nitrate increase and by growth delay of s.c. implanted colon 38 tumours. On the other hand, administration of the serotonin 5-hydroxytryptamine-2 (T-HT₂) antagonist cyproheptadine (20 mg/kg) inhibited both the plasma nitrate response and, to a lesser extent, the induction of tumour haemorrhagic necrosis by 5,6-MeXAA, FAA and TNF. Reduction of circulating plasma serotonin by pre-treatment withp-chlorophenylalanine and reserpine reduced the plasma nitrate response, but not the tumour necrosis response, to 5,6-MeXAA (30 mg/kg). It is suggested that serotonin is necessary for the induction of nitric oxide synthases and acts, either directly or indirectly, in concert with TNF. Serotonin agonists may have utility in increasing nitric oxide synthesis in response to TNF or to agents that induced TNF as part of their antitumour action.     

Haematological effects in mice of the antitumour agents xanthenone-4-acetic acid, 5,6-methyl-xanthenone-4-acetic acid and flavone acetic acid

Summary Treatment of C₅₇Bl/6×DBA/2 mice with the maximal tolerated dose of flavone-8-acetic acid (FAA, 1300 mol/kg), xanthenone-4-acetic acid (XAA, 1090 mol/kg), or its dose-potent derivative 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA, 100 mol/kg) resulted within 24 h in a dramatic reduction in the number of circulating lymphocytes, an elevation in haemoglobin concentrations and a reduction in platelet numbers. Neutrophil counts either remained unchanged or were slightly elevated. All three compounds caused a marked loss of cells in the thymus. Examination of histological sections of thymus at 48 h following treatment with XAA revealed a selective depletion of cortical thymocytes and no effects on the epithelium or other thymic structures. A transient decrease in cell numbers was seen in the spleen and femoral bone marrow, with recovery to normal levels occurring within 3 days. The number of haemopoietic stem cells, colony-forming units in culture (CFU-c), in the femoral bone marrow increased after drug administration despite the occurrence of a decrease in the overal number of cells in the femur. In contrast to the increase in CFU-c numbers seen in vivo, 2 h exposure of bone-marrow cells to FAA, XAA or 5,6-MeXAA in vitro resulted in a decrease in the surviving fraction of CFU-c. The results are consistent with the hypothesis that the in vivo haematological effects of these compounds are indirect, perhaps being mediated through the induction of cytokines, and contrast with the haematological effects of conventional antitumour agents. The biochemical and haematological effects are unlikely to be the cause of the acute toxicity observed for these compounds.This research was supported by the Auckland Division of the Cancer Society of New Zealand, the Medical Research Council of New Zealand, The Todd Foundation and a Warner-Lambert Laboratory Fellowship     

Evidence for the production of nitric oxide by activated macrophages treated with the antitumor agents flavone-8-acetic acid and xanthenone-4-acetic acid

Activated peritoneal macrophages, obtained from mice pretreated with Bacillus Calmette-Guérin, after exposure in vitro to flavone-8-acetic acid (FAA; NSC 347512) at a concentration of 890 microM, produce nitrite (3.7 nmol/10(6) cells), as measured 20 h later by the Griess reaction. Stimulation of nitrite production was inhibited at least 90% by NG-monomethylarginine (125 microM), suggesting that nitrite was formed via nitric oxide as a product of arginine metabolism. Stimulation was only partially inhibited by dexamethasone (0.1 microM). The ability of xanthenone-4-acetic acid (XAA) and three of its analogues to stimulate nitrite production was also investigated. 5,6-Dimethyl-XAA stimulated nitrite production (12.6 nmol/10(6) cells) at an optimal concentration of 80 microM, 8-methyl-XAA was without effect, and XAA and 5-methyl-XAA showed intermediate activity. The optimal in vitro drug concentrations for stimulation by FAA, XAA, and active XAA analogues correlated with the optimal in vivo dose required for the induction of either hemorrhagic necrosis or growth delay of s.c. Colon 38 tumors. These results strongly imply that FAA and active XAA derivatives function as low molecular weight stimulators of nitric oxide formation in macrophages, possibly acting on the same differentiation pathway as do endotoxin and tumor necrosis factor alpha. We suggest that nitric oxide, which is known to be toxic to tumor cells, contributes to the cytotoxic action of FAA and its analogues.     

Preclinical in vitro and in vivo activity of 5,6-dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid (5,6-MeXAA) is a fused tricyclic analogue of flavone acetic acid (FAA) which was developed in an attempt to improve on the activity of FAA. Previous studies have shown 5,6-MeXAA to be curative in 80% of mice bearing colon 38 tumours and 12 times more dose potent than FAA. This investigation has demonstrated that a murine colon tumour cell line (MAC15A) is approximately 60 times more sensitive to 5,6-MeXAA than to FAA, although these differences were not seen in three human cell lines tested. 5,6-MeXAA caused significant blood flow shutdown and haemorrhagic necrosis in subcutaneous MAC15A tumours in syngeneic and nude hosts, but measurable changes in tumour volume were seen only in syngeneic hosts. 5,6-MeXAA was inactive against intraperitoneal MAC15A but produced significant anti-tumour effects against the same cell line inoculated via an intravenous route. FAA has been shown previously to be inactive in this model. Interestingly, the effects against lung colonies were not accompanied by obvious necrotic changes, suggesting that they may be the result of increased direct cytotoxicity rather than an indirect host mechanism. Further studies to investigate the effects against systemic tumour deposits are under way.     

Persistent induction of nitric oxide synthase in tumours from mice treated with the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid.

An anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA) induced nitric oxide synthase (NOS) in the tumour, spleen, thymus and small intestine, but not in the lung, liver, kidney, heart or skeletal muscle in B6D2F1 mice bearing subcutaneous colon 38 tumours. This pattern of induction is distinct from that caused by agents such as endotoxin, muramyl dipeptide or Corynebacterium parvum. The induction of NOS (iNOS) in the tumour was more persistent (maximal at 3 days) than in other tissues (maximal at 12 h). Immunohistochemical staining suggested that iNOS was located in macrophages and endothelial cells within and around the tumour. Treatment with 5,6-MeXAA also caused substantial increases in plasma nitrite and nitrate (NOx) concentrations that peaked at 8-12 h after 5,6-MeXAA. The increase in plasma NOx was prevented by a NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), indicating that it was due to enhanced production of NO. Tumour-bearing mice were more responsive than controls to 5,6-MeXAA both in their plasma NOx increase and in their lower maximally tolerated dose. L-NIO was unable to prevent the complete tumour necrosis and regression caused by 5,6-MeXAA at a dose that substantially inhibited the increase of plasma NOx. In conclusion, the experimental anti-tumour agent 5,6-MeXAA induced NO synthesis in tumour-associated macrophages and in immunologically active tissues in parallel with its effects on tumour growth. The experiments with a non-selective NOS inhibitor L-NIO, however, suggest that NO is not a significant component in the mechanism of the anti-tumour action of 5,6-MeXAA in this particular model.     

Effect of thalidomide on tumour necrosis factor production and anti-tumour activity induced by 5,6-dimethylxanthenone-4-acetic acid.

The investigational anti-tumour agent, 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), an analogue of flavone acetic acid (FAA), has been scheduled for clinical evaluation. Like FAA, 5,6-MeXAA exhibits excellent experimental anti-tumour activity and is an efficient inducer of cytokines in mice. We have examined the effect of pharmacological suppression of tumour necrosis factor (TNF) production on the anti-tumour activity of 5,6-MeXAA, taking advantage of previous observations that TNF production in response to endotoxin in vitro is inhibited by thalidomide. Thalidomide at doses of between 8 and 250 mg kg-1 efficiently suppressed serum TNF activity in response to 5,6-MeXAA at its optimal TNF inducing dose of 55 mg kg-1. Suppression was achieved when thalidomide was administered at the same time as, or up to 4 h before, 5,6-MeXAA. Under conditions in which TNF activity was suppressed, the degree of tumour haemorrhagic necrosis and the proportion of cures in the subcutaneous Colon 38 tumour were increased. In mice administered thalidomide (100 mg kg-1) together with 5,6-MeXAA (30 mg kg-1), complete tumour regression was obtained in 100% of mice, as compared with 67% in mice receiving 5,6-MeXAA alone. The results suggest a possible new application for thalidomide and pose new questions about the action of 5,6-MeXAA and related compounds.     

Interaction between endotoxin and the antitumour agent 5,6-dimethylxanthenone-4-acetic acid in the induction of tumour necrosis factor and haemorrhagic necrosis of colon 38 tumours

The investigational antitumour agent 5,6-dimethyl-xanthenone-4-acetic acid (5,6-MeXAA) induced dosedependent haemorrhagic necrosis of colon 38 tumours to a similar extent to that induced using bacterial lipopolysaccharide (LPS). TNF- activity in serum and mRNA for TNF- in splenocytes were induced over a broad range of LPS doses, whereas with 5,6-MeXAA, induction occurred only at concentrations approaching the maximum tolerated dose. At concentrations that provided similar degrees of haemorrhagic necrosis, the levels of serum TNF- induced using 5,6-MeXAA were 100-fold lower than those obtained with LPS, indicating that haemorrhagic necrosis was not directly correlated with TNF- levels. There was also no correlation between the degree of tumour necrosis and the duration of growth delay. Treatment with LPS did not induce a singificant delay in growth, despite extensive tumour haemorrhagic necrosis, whereas with 5,6-MeXAA, treatments that improved the cure rate did not necessarily give longer growth delays. Therapy using a combination of sub-optimal doses of both compounds was synergistic for the induction of serum TNF- and message for TNF- but was not synergistic for antitumour efficacy. Thus, no correlation is evident between cure rates, duration of growth delay, haemorrhagic necrosis and TNF- induction by 5,6-MeXAA or LPS.     

Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells

Summary Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72%±20% survival of cells for epirubicin vs 45%±13% for doxorubicin at a concentration of 0.2 m;P0.005). As analyzed by the DiSC assay, 0.2 m epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival,P0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 m, the mean intracellular uptake of epirubicin in leukemic cells was 0.43±0.26 nmol/mg protein as compared with 0.33±0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 m was 0.47±0.25 nmol/mg protein as compared with 0.31±0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.     

Destruction of WiDr multicellular tumor spheroids with the novel thymidylate synthase inhibitor 1843U89 at physiological thymidine concentrations

The activity of a novel thymidylate synthase inhibitor, 1843U89, against WiDr human colon carcinoma multicellular tumor spheroids was investigated. Continuous exposure of the spheroids to 3 nM 1843U89 for 10 days resulted in spheroid disruption, whereas 100 nM methotrexate (MTX) was required for similar effects. Short-term treatment experiments demonstrated that a 3-day exposure to 100 nM 1843U89 caused spheroid disruption 9 days after drug removal. A 4-day exposure to 10 nM 1843U89 caused spheroid disruption 8 days after drug removal. In contrast, treatment with 10 or 100 nM 1843U89 for 6–48 h or treatment with 1 nM 1843U89 for up to 5 days caused only growth delay. Continuous exposure of spheroids to 30 nM 1843U89 in the presence of 0.05–0.3 M thymidine was as effective in causing spheroid disruption as treatment in the absence of thymidine, but treatment in the presence of 0.7–3.0 M thymidine caused partial reversal of spheroid disruption. The results of these experiments suggest that 1843U89 should have potent solid tumor activity in humans but should be less effective in mice due to differences in circulating thymidine levels (0.1 vs 1 M, respectively).     

Response to BCNU of spheroids grown from mixtures of drug-sensitive and drug-resistant cells

Summary Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R₃) to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). As shown previously, after treatment with 3M BCNU, percentages of each cell subpopulation in mixed-cell spheroids were estimated with the sister chromatid exchange (SCE) assay and found to be approximately the same as percentages used to initiate spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with BCNU, estimated by changes in the number of SCEs induced by treatment, decreased as the percentage of R₃ cells increased. When spheroids were disaggregated into single cells before treatment, however, the number of SCEs induced in the 9L population did not decrease but remained at levels similar to those found for spheroids grown from 9L cells only. These data suggest that the cell-cell interactions that influence BCNU sensitivity in mixed cell spheroids depend on three-dimensional intercellular contact. The response of purely 9L, purely R₃, and mixed-cell spheroids to BCNU was also determined using the cell survival and spheroid growth delay assays. The surviving fractions of individual spheroids treated with 40 M BCNU were slightly greater than expected; growth delays found for mixed-cell spheroids were 2–3 days less than expected. These findings suggest that cells in mixed-cell spheroids are more resistant to BCNU than would be predicted from the sensitivities of purely 9L and R₃ spheroids.Supported in part by NIH Grant CA-39465-02 and NIH program Project Grant CA-13525     

Pharmacodynamics and causes of dose-dependent pharmacokinetics of flavone-8-acetic acid (LM-975; NSC-347512) in mice

Summary Flavone acetic acid (FAA) is a novel antitumor agent with broad solid-tumor activity. However, this drug has shown a steep dose-response curve in preclinical trials, with a narrow sublethal window of efficacy. To investigate this threshold behavior, we studied various aspects of FAA pharmacology in mice after i.v. administration. Mice bearing advanced-stage s.c. colon 38 adenocarcinoma were treated at four dose levels (39, 65, 108, and 180 mg/kg), and only the highest dose produced significant antitumor activity, showing a steep dose-response curve. Using an HPLC assay, FAA pharmacokinetics in both plasma and tumors were found to be dose-dependent. As the dose increased, there was a decrease in both total body clearance and volume of distribution at steady state. The increase in tumor area under the curve (AUC) was more pronounced than the corresponding increase in plasma AUC, showing a better tumor exposure to FAA at high doses. The distribution of FAA in normal tissues showed a short-term retention in the liver and kidneys; low concentrations were observed in the heart, spleen, and brain, with some retention in the latter. The highest FAA concentrations were found in the gastrointestinal (GI) tract, mainly in the duodenum, suggesting an important biliary excretion of the drug. Various possible causes of FAA nonlinear pharmacokinetics were investigated. Serum protein binding was high (79%) and remained constant up to 100 g/ml, but decreased thereafter at higher FAA concentrations, e.g., 76% at 500 g/ml and 64% at 1,000 g/ml. Urinary and biliary clearances were dose-dependent and decreased 5- and 9-fold, from the 39- to the 180-mg/kg dose levels, respectively. A direct assessment of FAA enterohepatic circulation using intercannulated mice showed that 27% of the plasma AUC was accounted for by enterohepatic circulation. FAA acyl glucuronide was identified as the major metabolite in mice and was found to contribute to the nonlinear pharmacokinetics due to its facile hydrolysis under physiological conditions, regenerating FAA. In conclusion, the steep FAA dose-response curve was found to be caused by dose-dependent pharmacokinetics in mice. The nonlinear pharmacokinetics of this drug was attributed to a dose-dependent decrease in both urinary and biliary clearances, concentration-dependent serum protein binding, enterohepatic circulation, and the instability of FAA acyl glucuronide under physiological conditions, forming a futile cycle. The distribution data also suggested possible tissue targets for anticancer efficacy and/or toxicity that could be useful in designing clinical studies.This work was supported in part by the Wayne State University Ben Kasle Trust Fund for Cancer Research, by Public Health Service grants CA-43886 and CA-42449 from the National Cancer Institute, and by the Harper Medical Staff Trust Fund. Preliminary reports of this study were presented in abstract form [4, 8, 9]. Offprint requests to: Guy G. Chabot, Institut Gustave-Roussy, Pavillon de Recherche, 39 rue Camille Desmoulins, F-94805 Villejuif Cedex, France     

The use of vascularised spheroids to investigate the action of flavone acetic acid on tumour blood vessels

EMT6 multicellular spheroids were introduced into the peritoneal cavities of mice and allowed to become vascularised, resulting in solid spherical tumours. The necrotic cores of the initially avascular spheroids were replaced by vascularised tumour tissue but the outer zones of the spheroids failed to become vascularised. The presence of both vascular and avascular components in each spheroid allowed the role of the vasculature in the antitumour action of flavone acetic acid (FAA) to be determined. Eighteen hours after treatment with FAA 0.8 mmol kg-1, the vascularised core became necrotic and haemorrhagic, while the outer avascular zone remained viable. Tumour cells which were infiltrating superficial sub-mesothelial fat did not become necrotic despite the presence of numerous thrombi in associated vessels. Injection of two fluorescent vascular markers, the first (Hoechst 33342) together with FAA, and the second (10-nonyl acridine orange) 4 h later, demonstrated that there is a marked loss of blood flow in the spheroids. These results provide further evidence that FAA kills blood vessel-dependent tumour cells by interrupting the tumour blood supply.     

The role of immune effector cells in flavone acetic acid-induced injury to tumor cells in EMT6 spheroids.

Inhibition of tumor blood flow appears to a major antitumor mechanism of flavone acetic acid (FAA), although non-ischemic processes may also be a significant role. To distinguish between direct and immune effector cell-mediated cytotoxicity as the basis for non-ischemic killing, effects of FAA were compared in EMT6 spheroids grown entirely in vitro and spheroids recovered from the peritoneal cavities of mice after six days of in vivo growth (ex vivo spheroids). Approximately 50% of the cells in the latter case were of host origin (macrophages and lymphocytes). Ex vivo spheroids showed specific histological changes when exposed to FAA, including tumor cell rounding, apoptosis, depression of mitotic activity and dissolution of necrotic debris in the spheroid core. Quantitation of histological changes indicated these effects to be significantly greater in ex vivo than in vitro spheroids. The histological changes in FAA treated ex vivo spheroids were partially inhibited by dexamethasone. Oxygen tension did not influence the response of spheroids to FAA. The results suggest that immune effector cells, probably macrophages, mediate blood flow-independent antitumor effects of FAA.     

Tumor-dependent increased plasma nitrate concentrations as an indication of the antitumor effect of flavone-8-acetic acid and analogues in mice

The antitumor agent flavone-8-acetic acid (FAA) is remarkable because it induces hemorrhagic necrosis, altered tumor blood flow, and cytokine synthesis. We show here that FAA and structurally related analogues increase plasma nitrite plus nitrate (NO2-/NO3-) levels in mice. Dose-dependent increases in plasma NO2-/NO3- concentrations, which reached maximum levels at 12 h, were found following administration of FAA. Furthermore, the presence of a palpable s.c. Colon 38 tumor significantly enhanced the response. Tumor-dependent increases were also observed with the active FAA analogues xanthenone-4-acetic acid, 5-methyl XAA, and 5,6-dimethyl XAA, while the inactive analogue 8-methyl XAA failed to increase plasma NO2-/NO3- concentrations substantially above basal levels. Increased plasma NO2-/NO3- levels were also observed in response to endotoxin (100 micrograms/mouse) and to recombinant human tumor necrosis factor alpha (4 to 16 micrograms/mouse). NO2-/NO3- levels may signify nitric oxide production as a result of stimulation of the L-arginine-dependent pathway in activated macrophages. The tumor dependence of the response may reflect the immunological stimulus imposed by tumor implantation. A clear relationship was found between increased plasma NO2-/NO3- levels and tumor growth delays induced by FAA and xanthenone-4-acetic acid analogues. It is suggested that nitric oxide may contribute to tumor cell death by two mechanisms, alteration of blood flow contributing to tumor ischemia and direct tumor cell killing. Plasma NO2-/NO3- concentrations may be a sensitive indication of the antitumor response to this class of compounds.     

Flavone acetic acid (LM-975; NSC-347512) activation to cytotoxic species in vivo and in vitro

Summary Flavone acetic acid (FAA; LM 975; NSC 347512) is a new anticancer agent with unprecedented, broad antitumor activity in murine models. Although FAA is very effective in vivo against solid tumors, including colon 38 adenocarcinoma, it was not cytotoxic in vitro against colon 38 cells and human colon adenocarcinoma cells HCT116 at pharmacologically achievable concentrations and exposure times. For example, a concentration of 300 g/ml for a 10-day exposure time was required to obtain <1log cell kill. After the administration of an effective FAA dose (180 mg/kg, i.v.) to mice, plasma cytotoxicity against HCT116 cells attained a 2 log cell kill between 0.5 and 2 h, which decreased to 1 log cell kill at 4 h. No cytotoxicity was observed 6, 12 or 21 h after drug administration. The controls used comprised mouse plasma containing FAA concentrations similar to those assayed in the above plasma samples from in-vivo-dosed mice. These apiked plasma were not cytotoxic, indicating that other cytotoxic species, formed in vivo, were responsible for the increased cytotoxicity. Mouse hepatocytes co-cultured with HCT116 cells increased FAA cytotoxicity to 1 log cell kill at 30–100 g/ml. The addition of phenobarbital-induced mouse liver supernatant S-9000xg also markedly increased FAA cytotoxicity to a 2 log cell kill at 300 g/ml. We conclude that FAA can be activated both in vivo and in vitro to cytotoxic species that are more active than the parent compound.Part of this work has been published in abstract form in: Proc Am Assoc Cancer Res 29: 484, 1988     

Pharmacokinetics of PEG-l-asparaginase and plasma and cerebrospinal fluidl-asparagine concentrations in the rhesus monkey

The pharmacokinetics of the polyethylene glycol-conjugated form of the enzymel-asparaginase and the depletion ofl-asparagine from the plasma and cerebrospinal fluid (CSF) following an i.m. dose of 2500 IU/m² PEG-l-asparaginase was studied in rhesus monkeys. PEG-l-asparaginase activity in plasma was detectable by 1 h after injection and maintained a plateau of approximately 4 IU/ml for more than 5 days. Subsequent elimination from plasma was monoexponential with a half-life of 6±1 days. Plasmal-asparagine concentrations fell from pretreatment levels of 14–47 M to <2 M by 24 h after injection in all animals and remained undetectable for the duration of the 25-day observation period in four of six animals. In two animals, plasmal-asparagine became detectable when the PEG-l-asparaginase plasma concentration dropped below 0.1 IU/ml. Pretreatment CSFl-asparagine levels ranged from 4.7 to 13.6 M and fell to <0.25 M by 48 h in five of six animals. CSFl-asparagine concentrations remained below 0.25 M for 10–14 days in four animals. One animal had detectable CSFl-asparagine concentrations within 24 h and another had detectable concentrations within 1 week of drug administration despite a plasma PEG-l-asparaginase activity profile that did not differ from that of the other animals. These observations may be useful in the design of clinical trials with PEG-l-asparaginase in which correlations among PEG-l-asparaginase pharmacokinetics, depletion ofl-asparagine, and clinical outcome should be sought.     

Anti—vascular approaches to solid tumour therapy: Evaluation of vinblastine and flavone acetic acid

Several agents have now been identified which exert their anti-tumour effects in large part via the tumour vasculature; these include TNFα and flavone acetic acid (FAA). More recently, Vincristine and Vinblastine have also been shown to cause a prolonged and selective decrease in tumour perfusion. Vinblastine, unlike FAA, causes no increase in plasma TNFα levels in mice bearing the CaNT tumour, suggesting 2 distinct mechanisms of anti-vascular activity for these structurally diverse agents. Since FAA and Vinblastine also show quite different normal tissue toxicities, which are separately dose-limiting, we have examined the strategy of combining these 2 agents. When Vinblastine preceded FAA by 24 hr or less, tumour growth delay was significantly enhanced without a concomitant increase in toxicity. The level of enhancement was not significantly reduced by a 5-fold decrease in Vinblastine dose, though any reduction in the dose of FAA caused a rapid reduction in treatment effectiveness. Investigation of the functional vasculature of treated tumours suggested that increased anti-vascular effects may contribute to the enhanced growth inhibition of the combined treatment. Our results demonstrate the potential benefit of combining 2 different classes of antivas-cular agent, using Vinblastine and FAA (or 5,6-MeXAA) as prototype drugs.     

Dose-dependent pharmacokinetics of flavone acetic acid in mice

Summary The pharmacokinetics of the novel anticancer agent, flavone acetic acid (FAA) were investigated in Balb-c mice treated with i. v. doses of 100 mg/kg or 300 mg/kg, using an HPLC assay. The kinetics of disappearance from plasma was monoexponential and dose-dependent. After 100 mg/kg, the plasma peak level was 250±11 g/ml, t^1/2 was 0.5 h, and AUC was 309 g/ml per h. After 300 mg/kg, the plasma peak level was 710±57 g/ml, t^1/2 was 2.1 h, and the area under the curve (AUC) 1771 g/ml h. Mouse plasma protein binding of FAA was about 70%. As is the case with plasma, in all tissues analyzed, the FAA-AUC values were disproportinately greater after 300 mg/kg than after 100 mg/kg. The highest drug concentrations were found in the liver and small intestine; concentrations were intermediate in lung, heart, and spleen, and lowest in brain. Less than 5% of the FAA dose was eliminated as unchanged drug in the stool. Total excretion of FAA as unchanged drug in the urine collected up to 96 h after drug treatment corresponded to 75% and 60% of the i. v. doses of 100 and 300 mg/kg, respectively. A minor fraction of FAA dose, corresponding to 1% and 6% of the two doses, was eliminated in the urine as a FAA glucuronide or sulfate.