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.     

Nitric oxide: its production in host-cell-infiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

Summary Flavone-8-acetic acid (FAA) and its more dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), appear to exert their antitumour effects through vascular and other host-mediated mechanisms and are known to induce the synthesis of nitric oxide by murine macrophages. We investigated the role of nitric oxide in the cytotoxic effects of these drugs in host-cell-infiltrated spheroids. EMT6 murine mammary adenocarcinoma cells were grown in culture to produce multicellular spheroids in vitro spheroids), which were then inoculated i. p. into mice. After 6 days the spheroids were removed ex vivo spheroids). Exposure to FAA (890 m) and 5,6-MeXAA (80 m) in vitro for 20 h increased nitrite concentrations to 6.7 and 9.7 nmol/spheroid, respectively, as compared with 0.7 nmol/spheroid in the absence of drug. FAA and 5,6-MeXAA did not increase nitrite production in in vitro spheroids in cells obtained by peritoneal lavage. However, mixed cultures of in vitro spheroids and peritoneal cells treated with 5,6-MeXAA produced nitrite (2.5 nmol/spheroid), indicating that interactions between host cells and tumour cells were important for induction. The effets of these drugs on ex vivo spheroids were prevented by co-incubation withN ^G-monomethyl-l-arginine, indicating that nitrite originated from the oxidation ofl-arginine to nitric oxide. Cell sorting of disaggregated spheroids into EMT6 cells andMac-1-positive macrophage populations indicated that both of these cell populations could be induced to synthesise nitric oxide by subsequent incubation with 5,6-MeXAA. Incubation of ex vivo spheroids with FAA and 5,6-MeXAA decreased the clonogenicity of EMT6 cells, and this effect was wholly (FAA) or partially (5,6-MeXAA) reversed by the presence ofN ^G-monomethylarginine (250 m). FAA and 5,6-MeXAA may therefore exert some of their cytotoxic effects on tumour cells through the production of nitric oxide.This work was supported by grants from the New Zealand Lotteries Board, the Cancer Society of New Zealand and the Health Research Council of New Zealand     

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     

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.     

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.     

Oral activity and pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice

PURPOSE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an anticancer drug with an antivascular action, has recently completed phase I clinical trials. Since oral administration has many advantages, we compared the biological activity and pharmacokinetics of DMXAA in mice following oral and intraperitoneal (i.p.) administration. METHODS: Growth delays of Colon 38 tumours were measured in C57Bl/6 mice. Plasma concentrations of DMXAA, 5-hydroxyindole-3-acetic acid (5HIAA) as a measure of serotonin production, and nitrate as a measure of nitric oxide production, were determined by high-performance liquid chromatography. Tumour necrosis factor (TNF) concentrations in serum and tumour tissues were measured by ELISA. RESULTS: The antitumour activity of DMXAA at the maximum tolerated oral dose (32.5 mg/kg) was low (4-day growth delay, no cures) compared to that (19-day growth delay, 40% cures) at the maximum tolerated i.p. dose (27.5 mg/kg). The pharmacokinetics of DMXAA in plasma, liver and tumour tissue indicated a bioavailability of 73%. Elevation of plasma 5HIAA, measured 4 h following i.p. administration of DMXAA, was linear with DMXAA dose, and the 5HIAA response to oral administration was consistent with its bioavailability. TNF concentrations increased following oral administration (30 mg/kg) and were particularly evident in tumour tissue, but were lower and less prolonged than those in response to i.p. administration at 25 mg/kg. Plasma nitrate levels were not increased following oral administration (30 mg/kg). CONCLUSIONS: DMXAA exhibits good bioavailability, and changes in serum TNF, tissue TNF, plasma 5HIAA and plasma nitrate, as markers of biological response, are consistent with this bioavailability. The low maximal plasma DMXAA concentration following oral administration, resulting in reduced retention of intratumoral TNF, may be responsible for the low antitumour activity.     

The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF receptor-1 knockout mice

5,6-dimethylxanthenone-4-acetic acid, a novel antivascular anticancer drug, has completed Phase I clinical trial. Its actions in mice include tumour necrosis factor induction, serotonin release, tumour blood flow inhibition, and the induction of tumour haemorrhagic necrosis and regression. We have used mice with a targeted disruption of the tumour necrosis factor receptor-1 gene as recipients for the colon 38 carcinoma to determine the role of tumour necrosis factor signalling in the action of 5,6-dimethylxanthenone-4-acetic acid. The pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid, as well as the degree of induced plasma and tissue tumour necrosis factor, were similar in tumour necrosis factor receptor-1(-/-) and wild-type mice. However, the maximum tolerated dose of 5,6-dimethylxanthenone-4-acetic acid was considerably higher in tumour necrosis factor receptor-1(-/-) mice (>100 mg kg(-1)) than in wild-type mice (27.5 mg kg(-1)). The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (25 mg kg(-1)) was strongly attenuated in tumour necrosis factor receptor-1(-/-) mice. However, the reduced toxicity in tumour necrosis factor receptor-1(-/-) mice allowed the demonstration that at a higher dose (50 mg kg(-1)), 5,6-dimethylxanthenone-4-acetic acid was curative and comparable in effect to that of a lower dose (25 mg kg(-1)) in wild-type mice. The 5,6-dimethylxanthenone-4-acetic acid -induced rise in plasma 5-hydroxyindoleacetic acid, used to reflect serotonin production in a vascular response, was larger in colon 38 tumour bearing than in non-tumour bearing tumour necrosis factor receptor-1(-/-) mice, but in each case the response was smaller than the corresponding response in wild-type mice. The results suggest an important role for tumour necrosis factor in mediating both the host toxicity and antitumour activity of 5,6-dimethylxanthenone-4-acetic acid, but also suggest that tumour necrosis factor can be replaced by other vasoactive factors in its antitumour action, an observation of relevance to current clinical studies.     

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     

Effects of the serotonin receptor antagonist cyproheptadine on the activity and pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

BACKGROUND: DMXAA (5,6-dimethylxanthenone-4-acetic acid) is a new drug synthesized in this laboratory and currently in phase I clinical trial. In mice it acts as an antivascular drug, selectively inhibiting tumour blood flow and inducing tumour haemorrhagic necrosis with resultant tumour regression. It also induces the synthesis of tumour necrosis factor (TNF), nitric oxide and serotonin. Cyproheptadine, a type 2 serotonin receptor antagonist, is known to reduce the degree of tumour necrosis-induced TNF in mice. We investigated the pharmacological interaction between a suboptimal dose of DMXAA (20 mg/kg) and cyproheptadine (20 mg/ kg) using mice with Colon 38 tumours that are sensitive to DMXAA. METHODS: Mice with or without tumours were treated with DMXAA and/or cyproheptadine. Concentrations of plasma and tissue DMXAA and the serotonin metabolite 5-hydroxyindoleacetic acid were measured by high performance liquid chromatography. TNF concentrations were measured by ELISA. RESULTS: While DMXAA alone (20 mg/kg) showed little or no antitumour activity, coadministration with cyproheptadine was curative in four of five mice. DMXAA half-lives in plasma and tumour tissue were increased 5.1- and 5.6-fold, respectively, and the appearance of DMXAA glucuronides in bile was almost completely inhibited for up to 4 h. Serum TNF was low and unchanged by cyproheptadine, and plasma concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also not substantially changed. CONCLUSION: The augmentation by cyproheptadine of the induction of tumour response to DMXAA reflects a pharmacological interaction, leading to increased plasma and tumour half-lives, and to reduced excretion. However, serum TNF concentrations were not increased, suggesting that the increased anti-tumour effects are mediated by an increased local tumour response, arising from the extended tumour DMXAA concentrations.     

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.     

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.     

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.     

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.     

Induction of natural killer activity by Xanthenone analogues of flavone acetic acid: Relation with antitumour activity

Flavone-8-acetic acid (FAA) induces haemorrhagic necrosis and tumour regression in experimental tumours and induces natural killer (NK) activity. Xanthenone-4-acetic acid (XAA) forms the basis of a series of analogues of FAA which vary in antitumour potency. FAA, XAA and 15 XAA derivatives were tested for their ability to induce either NK activity in mouse spleens or haemorrhagic necrosis in mouse colon 38 tumours. Some derivatives were active in both assays (one at a dose 8-fold lower than that of FAA). When both assays were quantitated, a significant correlation (r = 0.85; P < 0.001) was found. NK assays could be useful in screening compounds such as FAA and XAA analogues which appear to mediate their antitumour activity by biological response modification. Since tumour necrosis may not be mediated directly by NK cells, FAA and active XAA derivatives may exert pleiotropic effects that include NK induction and tumour necrosis by acting on host cells to release cytokines.     

Mechanisms of enhancement of the antitumour activity of melphalan by the tumour-blood-flow inhibitor 5,6-dimethylxanthenone-4-acetic acid

 Several studies show that the antitumour activity of melphalan (MEL) and other alkylating agents can be enhanced by the selective inhibition of tumour blood flow, although the mechanism(s) underlying these interactions are unclear. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer agent currently in phase I clinical trial, inhibits blood flow in murine tumours. DMXAA increased the activity of MEL against the MDAH-MCa-4 mouse mammary tumour maximally when MEL was given about 2 h after DMXAA, without compromising the maximal dose of the alkylating agent that could be given. The plasma pharmacokinetics of MEL were unchanged by DMXAA pretreatment, but the area under the concentration-time curve (AUC) for the tumour increased by 33% as a result of decreasing clearance (consistent with falling tumour blood flow). However, inhibition of tumour blood flow also leads to microenvironmental changes (e.g. acidosis and hypoxia) that might influence sensitivity to MEL. The sensitivity of KHT cells (freshly isolated from tumours) to MEL in vitro was increased by lowering of either pH or oxygen concentration (pO₂), with an overall dose-modifying factor of 15 being recorded for aerobic cells at pH 7.4 versus hypoxic cells at pH 6.5. The cellular uptake of MEL by KHT cells was increased by 74% under hypoxia. Thus, This study was supported by the National Cancer Institute, USA (contract NO1-CM-47019), and the Health Research Council of New Zealand DMXAA appears to augment the antitumour activity of MEL through two different mechanisms, increased exposure (via decreased tumour clearance of MEL) and increased sensitivity resulting from changes to the tumour microenvironment, both of which result from inhibition of tumour blood flow. Received: 13 July 1996 / Acepted: 4 November 1996     

Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis

Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor- (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C₃H and BDF₁ (C₅₇BL6×DBA₂) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was converted rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrite, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71±0.04 l/kg and the clearance was 0.32±0.02 l/h^–1/kg^–1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.     

Production of tumour necrosis factor-alpha by cultured human peripheral blood leucocytes in response to the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid (DMXAA, NSC 640488), developed in this laboratory as an improved analogue of flavone acetic acid (FAA, NSC 347512), is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.     

Induction of endothelial cell apoptosis by the antivascular agent 5,6-Dimethylxanthenone-4-acetic acid

5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg x kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg x m(-2)). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg x m(-2)). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction.     

DMXAA: an antivascular agent with multiple host responses

: To measure host responses to the antivascular agent DMXAA (5,6-dimethylxanthenone-4-acetic acid) and to compare them with those of other antivascular agents.

: Induction of tumor necrosis was measured in s.c. murine Colon 38 carcinomas growing in normal or tumor necrosis factor (TNF) receptor-1 knockout mice. Plasma and tumor tissue TNF concentrations were measured by ELISA. Plasma concentrations of 5-hydroxyindoleacetic acid (as a measure of serotonin release) and nitrite (as a measure of nitric oxide release) were measured by high-performance liquid chromatography.

: Administration of DMXAA to tumor-bearing mice increased plasma and tumor tissue-associated TNF, in addition to increasing plasma nitric oxide, distinguishing its action from that of mitotic poisons that had an antivascular action. Results from TNF receptor-1 knockout mice showed that TNF played an important role in both its antitumor action and its host toxicity. Release of serotonin occurred in response to mitotic poisons, as well as to DMXAA.

: The antivascular action of DMXAA involves in situ production in tumor tissue of a cascade of vasoactive events, including a direct effect on vascular endothelial cells and indirect vascular effects involving TNF, other cytokines, serotonin, and nitric oxide. Now that Phase I clinical trials of DMXAA are completed, the optimization of this cascade in cancer patients is a major challenge. Plasma 5-hydroxyindoleacetic acid concentrations may provide a useful surrogate marker for the antivascular effects of DMXAA and other antivascular agents.