Aims: DMXAA (AS1404), a small-molecule vascular disrupting agent that has now completed Phase II clinical trial, induces endothelial cell apoptosis, increased vascular permeability and decreased tumour blood flow in vivo. Its action is incompletely understood and we wished to develop an in vitro system to study its effects.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) (AS1404) is a novel antitumour agent that selectively disrupts tumour vasculature and induces cytokines. The purpose of this study was to determine the pharmacokinetics (PK) of DMXAA in cancer patients enrolled in a phase I clinical trial.

3123 Background: VTAs now undergoing clinical trials include those that interact with tubulin (e.g. combretastatins and ZD6126) and the distinct tubulin-independent based flavonoid DMXAA (AS1404). DMXAA induces both direct apoptosis of tumor vascular endothelial cells and a secondary induction of vasoactive agents such as serotonin (from platelets aggregating to damaged vasculature) and tumor necrosis factor (TNFα). The study objective was to determine whether plasma levels of the serotonin metabolite, 5HIAA, correlated with DMXAA induced blood flow changes, first in mouse models and then in patients. Methods: 5HIAA levels from blood, were determined by HPLC. Mice, bearing syngeneic colon 38 subcutaneous tumors, were given single doses of DMXAA (up to ∼150mg/m2). Within a recently completed Phase I double-blind randomised study in refractory tumors; DART) patients received 20 minute intravenous infusions of DMXAA at 300 to 3000 mg/m2. Results: 5HIAA levels in mice measured 4hr post DMXAA showed a significant linear correlation with increased extravasation of the albumin binding Evans Blue from tumors (r=0.82; P<0.05); extravasation significantly correlated with reduced tumor blood flow (r = 0.88; P<0.01). In the same mice, no change in extravasation was seen in normal skin. In patients, peak 5HIAA plasma levels occurred at 4hr post dosing at dose levels >600mg/m2. Notably, there was a positive correlation between 5HIAA plasma levels and DMXAA dose up to 1200mg/m2 but thereafter a plateau was observed (even though plasma levels of free DMXAA increased linearly with dose up to 3000mg/m2). Conclusions: Increased plasma levels of 5HIAA may represent a sensitive biological marker of blood flow changes induced by the VTA, DMXAA. Dose-response data from Phase I trial patients receiving DMXAA show that the optimum biological dose to cause tumor blood flow/5HIAA changes is in the range of 1200mg/m2, that is, well below the maximum tolerated dose. Consequently, doses in this range are being studied in Phase II combination trials with taxanes (where marked synergy was seen in various preclinical tumor models) and platins. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antisoma Antisoma

The novel vascular targeting agent 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) has completed phase 1 clinical trial and has shown tumor antivascular activity in both mice and humans. We have investigated its ability to change tumor vascular permeability, relating it to tumor vascular perfusion and other responses. The murine colon 38 adenocarcinoma was grown in C57Bl wild‐type mice and mice lacking expression of either tumor necrosis factor receptor‐1 (TNFR1−/−) or TNF (TNF−/−). Tumor vascular permeability, as measured by extravasation of albumin‐Evans Blue complexes 4 hr after DMXAA treatment, was significantly increased in tumor tissue in C57Bl, TNFR1−/− and TNF−/− mice but not in normal (skin) tissue. Significant linear relationships were found between increased tumor vascular permeability, decreased functioning tumor blood vessels (measured by Hoechst 33342 staining at 4 hr), increased plasma 5‐hydroxyindole‐3‐acetic acid concentrations (as a measure of serotonin release by platelets) and the degree of induced tumor hemorrhagic necrosis. The results support the hypothesis that DMXAA increases tumor vascular permeability both directly and through the induction of other vasoactive mediators, including TNF. DMXAA might be useful clinically to potentiate the vascular permeability of other anticancer modalities such as cytotoxic drugs, antibodies, drug conjugates and gene therapy. © 2005 Wiley‐Liss, Inc.

PURPOSE:5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer drug that has recently completed Phase I clinical trial, is effective against transplantable murine tumors with established vasculature. We wished to determine the relationship between administration schedule and antitumor activity.EXPERIMENTAL DESIGN:C57Bl/6 mice with s.c. implanted Colon 38 tumors were used for determination of maximal tolerated doses and tumor growth delay. Plasma and tissue DMXAA concentrations were measured by high-performance liquid chromatography.RESULTS:Continuous infusion (30 mg/kg/day for 3 days) and daily i.p. administration schedules (7.5 mg/kg) were ineffective. A pharmacokinetically guided schedule was developed to increase tumor tissue drug concentrations without increasing the maximal plasma concentration. A schedule comprising a loading dose (25 mg/kg, i.p.) followed by supplementary doses (5 mg/kg after 4 and 8 h) provided a 1.6-fold increase in tumor tissue area under the concentration-time curve, no increased toxicity, and superior antitumor activity (100% cure rate, as compared with 55% for a single i.p. dose of 25 mg/kg). A similar strategy was developed for oral administration with a loading dose (30 mg/kg) and supplementary doses (15 mg/kg after 4 and 8 h). It provided a 90% cure rate, in contrast to a single oral dose (0% cure rate).CONCLUSIONS:The antitumor action of DMXAA is schedule dependent, and the achievement of an adequate tumor tissue DMXAA concentration above a threshold value appears to be critical for activity. The use of a pharmacokinetically guided schedule provides excellent oral activity against Colon 38 tumors and provides a basis for developing more effective administration schedules in clinical trials.

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.

. 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.

Background: Serotonin (5HT), a naturally occurring vasoactive substance, is released from platelets into plasma under various pathological conditions. Recently, anticancer drugs that act by selectively disrupting tumour blood flow have been found to increase plasma 5HT concentrations in mice. Two such antivascular agents, flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have completed Phase I clinical trial and raise the important question of whether suitable surrogate markers for antivascular effects can be identified. Methods: 5HT is unstable to storage, precluding routine clinical assay, but the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) accumulates in plasma following 5HT release and is a more suitable marker because of its greater stability. We have developed an automated procedure for the assay of the low concentrations of 5HIAA found in humans by combining solid-phase extraction with high-performance liquid chromatography (HPLC). Results: Efficient separation of 5HIAA from possible interfering substances in human plasma, including a variety of pharmaceutical agents, was achieved on C18 columns using cetyltrimethylammonium bromide (CETAB) as an organic modifier. Adequate precision, accuracy and sensitivity were achieved by electrochemical detection (ECD) at +400 mV. Analysis of plasma from two patients treated with DMXAA in a Phase I trial demonstrated DMXAA-induced elevation of plasma 5HIAA with a time course similar to that previously described in mice. Conclusions: Measurement of changes in plasma 5HIAA provides a new approach to the monitoring of therapies with an antivascular effect. The assay is sensitive to dietary sources of 5HT, which should be minimised.

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.

Background: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an investigative drug currently in clinical trial, acts on tumour vasculature through the induction of cytokines. Coadministration of thalidomide, a modulator of cytokine production, potentiates the antitumour activity of DMXAA against the murine Colon 38 carcinoma in mice. We wished to determine whether alteration of the pharmacokinetics of DMXAA by thalidomide could provide an explanation for this potentiation. Results: Coadministration of thalidomide to Colon 38 tumour-bearing mice significantly (P < 0.05) increased the elimination half-life (t1/2) of DMXAA in plasma (413 μmol/l), liver (132 μmol/l), and spleen (77 μmol/l), and significantly (P < 0.05) increased DMXAA concentrations in Colon 38 tumour tissue (0.25–4.5 h). l-Thalidomide had a greater effect on DMXAA elimination (P < 0.01) than did d-thalidomide or the racemate. Coadministration of thalidomide increased the area under the concentration-time curve (AUC) of DMXAA by 1.8-fold in plasma, liver and spleen, and by 3.0-fold in tumour. Bile from mice given thalidomide and DMXAA contained substantially lower amounts of the glucuronide metabolite of DMXAA (DMXAA-G) than did bile from mice given DMXAA alone. Conclusion: Glucuronidation is a major excretory pathway for DMXAA in the mouse. Thalidomide, probably as the l-form, decreases the rate of elimination of DMXAA from plasma, spleen, liver and tumour by altering the rate of glucuronidation. The reduction in the elimination of DMXAA by thalidomide may lead to a selective increase in exposure of tumour tissue to drug, providing a basis for its potentiation of antitumour activity.