Drug 5,6-dimethylxanthenone-4-acetic Acid Research Articles

Non-specific binding of the experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in liver microsomes from various species.

Total (added) drug concentrations other than unbound concentrations have been used to estimate the in-vitro enzyme kinetic parameters for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an experimental anti-cancer drug. This study aimed to investigate the non-specific binding of DMXAA to liver microsomes from variousspecies and to microsomesfrom human lymphoblastoid cells expressing drug-metabolising enzymes, and to examine the effect of the binding on the estimation of enzyme kinetic parameters for DMXAA in-vitro. The separation of unbound DMXAA was conducted by ultrafiltration and DMXAA concentrations were determined by validated HPLC. The results indicated that DMXAA was bound to liver microsomes and lymphoblastoid cell microsomes to a small extent (free fraction in microsomes, f(u(mic)) mostly > 0.85). Correction forthe unbound DMXAA concentration resulted in slightly lower apparent Michaelis-Menten constant (Km) values, but with the maximal velocity of reaction (Vmax) unchanged, leading to slightly higher unbound Vmax/Km values. These results indicate that the non-specific binding of DMXAA to microsomes is insignificant and has little impact on the enzyme kinetic estimation in-vitro.

5,6‐Dimethylxanthenone‐4‐acetic acid treatment of a non‐immunogenic tumour does not synergize with active or passive CD8+ T‐cell immunotherapy

The chemotherapeutic drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) inhibits intratumoural blood flow, causing hypoxia, haemorrhagic necrosis, vascular collapse and tumour cell death. Production of TNF-alpha and IFN is also induced, causing local inflammation and activation of immune cells including CD8+ T cells. We used the tumour cell line LL-LCMV, which expresses the gp33 epitope of lymphocytic choriomeningitis virus in a non-immunogenic form, to investigate whether tumour cell death caused by treatment with DMXAA may improve the success of tumour immunotherapy mediated by CD8+ T cells. Treatment with DMXAA was effective at reducing the size of LL-LCMV tumours. However, compared to normal mice, tumour reduction was no more marked or sustained in mice carrying high numbers of naive, tumour-specific CD8+ T cells. The antitumour effect of activated CD8+ T cells was also not affected by DMXAA treatment. Tumour-specific CD8+ T cells activated in vivo by immunization with dendritic cells and specific tumour peptide antigen, or generated in vitro and adoptively transferred into tumour-bearing mice by i.v. injection, did not improve or sustain the reduction in tumour size induced by DMXAA treatment. We conclude that the presence of high numbers of naive CD8+ T cells, or immunotherapies leading to CD8+ T-cell activation, do not synergize with the tumour cell death and local inflammation induced by DMXAA treatment. It is possible that this lack of synergism may result from both treatments inducing activation of CD8+ T cells and that treatments that activate different populations of immune cells may achieve better success.

138 Enhancement of the action of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by co-administration of non-steroidal anti-inflammatory drugs

138 Enhancement of the action of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by co-administration of non-steroidal anti-inflammatory drugs

149 Inhibition of vasculogenic mimicry in melanoma by the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

149 Inhibition of vasculogenic mimicry in melanoma by the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

Requirements for ICAM-1 immunogene therapy of lymphoma.

Intercellular cell adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein capable of eliciting bidirectional signals that activate signalling pathways in leukocytes, endothelial, and smooth muscle cells. Gene transfer of xenogeneic ICAM-1 into EL-4 lymphomas causes complete tumor rejection; however, it is unknown whether the mechanism responsible involves the "foreignness" of the ICAM-1 transgene, bidirectional signalling events, ICAM-1-receptor interaction, or a combination of the latter. To begin to address this question, we constructed four different therapeutic expression vectors encoding full-length ICAM-1, and forms in which the N-terminal ligand-binding domains and cytoplasmic tail had been deleted. Mouse EL-4 tumors (0.5 cm in diameter), which actively suppress the immune response, were significantly inhibited in their growth following injection of expression plasmids encoding either full-length xenogenic (human) ICAM-1, or a functional cytoplasmic domain-deficient form that retains ligand-binding activity. Efficacy of ICAM-1-mediated antitumor immunity was significantly augmented by administration of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which suppressed blood supply to the tumor, leading to enhanced leukocyte infiltration, and complete tumor eradication in a gene dosage and CD8(+) T cell and NK cell-dependent fashion. Generation of potent cytotoxic T cell (CTL)-mediated antitumor immunity was reflected by ICAM-1-facilitated apoptosis of tumor cells in situ. In contrast, nonfunctional ICAM-1 lacking the N-terminal ligand-binding Ig domain failed to generate antitumor immunity, even in the presence of DMXAA. These studies demonstrate that ICAM-1-stimulated antitumor immunity can overcome tumor-mediated immunosuppression, particularly when employed in combination with an attack on the tumor vasculature. The ligand-binding domain of ICAM-1 is essential for generating antitumor immunity, whereas the cytoplasmic domain and bidirectional activation of tumor signalling pathways are not essential.

Preclinical factors affecting the interindividual variability in the clearance of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid

Cancer chemotherapy is characterized by significant interindividual variations in systemic clearance, therapeutic response, and toxicity. These variations are due mainly to genetic factors, leading to alterations in drug metabolism and/or target proteins. The aim of this study was to determine, using a human liver bank (N=14), the interindividual variations in the expression and activity of liver enzymes that metabolize the investigational anticancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), i.e cytochrome P450 (CYP1A2) and uridine diphosphate glucuronosyltransferase (UGT1A9/2B7). In addition, interindividual variations in enzyme inhibition, hydrolysis of DMXAA acyl glucuronide (DMXAA-G) by plasma and hepatic microsomes, and the binding of DMXAA by plasma proteins also were examined. The results indicated that there was approximately one order of magnitude of interindividual variation in the expression of CYP1A2 and UGT2B7, activity of the enzymes toward DMXAA, and inhibition potency ( ic 50) by diclofenac, cyproheptadine, and α-naphthoflavone. The enzyme activities toward DMXAA and ic 50 values were closely correlated with enzyme expression. There was a smaller (2- to 3-fold) variation in the enzyme-catalyzed hydrolysis of DMXAA acyl glucuronide in human plasma and liver microsomes (N=6) and in the binding of DMXAA by plasma proteins in humans. In conclusion, the interindividual variability of DMXAA disposition observed in vitro might reflect the greater elimination variability (>one order of magnitude) in Phase I cancer patients. The variability in DMXAA clearance in these cancer patients would be due mainly to differences in its metabolism and its metabolic inhibition by co-administered drugs. To a lesser extent, variability in the clearance of DMXAA could be due to the hydrolysis of its acyl glucuronide and/or its binding to plasma proteins. Further study is needed to examine the genotype–phenotype relationship, and the result, together with therapeutic drug monitoring may provide a useful strategy for optimizing DMXAA treatment.

Preclinical factors influencing the relative contributions of Phase I and II enzymes to the metabolism of the experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid

It is important to determine the relative contribution of each metabolic pathway ( f p ) and of enzymes to the net metabolism of a drug. The aim of this study was to investigate, using a human liver bank, the f p of the anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and the effects of various inhibitors and inducers on f p . The mean apparent K m and V max values (N=14) were 21±5 μM and 0.04±0.02 nmol/min/mg, respectively, for 6-methylhydroxylation, and 143±79 μM and 0.71±0.52 nmol/min/mg, respectively, for acyl glucuronidation in human liver microsomes. 6-Methylhydroxylation and acyl glucuronidation contributed 26 and 74%, respectively, to DMXAA metabolism at 5 μM; values were 7 and 93% at 350 μM DMXAA. There was a significant relationship between the ratio of metabolic activity by Phase II and I reactions ( R II/I) and uridine diphosphate glucuronosyltransferase (UGT2B7) protein level ( r=0.605, P=0.022), whereas a reverse correlation between R II/I and cytochrome P450 (CYP1A) protein level was observed ( r=−0.540, P=0.046). Various compounds inhibited either DMXAA glucuronidation or 6-methylhydroxylation, or both pathways. Pretreatment of rats with β-naphthoflavone, but not phenobarbitone and cimetidine, increased the percentage of the contribution by 6-methylhydroxylation to 17% from 4% of control at 5 μM DMXAA. Our results indicate that the f p of DMXAA is subject to substrate concentration, inhibition, induction, and the protein levels of enzymes that biotransform DMXAA. However, clinical studies are important to verify the conclusions drawn from in vitro data.

6-methylhydroxylation of the anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by flavin-containing monooxygenase 3.

The involvement of flavin-containing monooxygenase (FMO) in the 6-methylhydroxylation of the experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was investigated by use of human liver microsomes and microsomes containing cDNA-expressed FMOs. The involvement of FMO in the formation of 6-methyl hydroxylate of DMXAA, 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes was indicated by the fact that this biotransformation was sensitive to heat treatment, increased at pH 8.3, and inhibited by methimazole. Only FMO3 formed 6-OH-MXAA at a similar rate to that in cDNA-expressed cytochromes P-450 (CYP)1A2. The results of this study indicate that human FMO3 has the capacity to form 6-OH-MXAA, but plays a lesser important role for this reaction than CYP1A2 that has been demonstrated to catalyse 6-OH-MXAA formation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA): a new biological response modifier for cancer therapy.

The investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was developed by the Auckland Cancer Society Research Centre (ACSRC). It has recently completed Phase I trials in New Zealand and UK under the direction of the Cancer Research Campaign's Phase I/II Clinical Trials Committee. As a biological response modifier, pharmacological and toxicological properties of DMXAA are remarkably different from most conventional chemotherapeutic agents. Induction of cytokines (particularly tumour necrosis factor (TNF-alpha), serotonin and nitric oxide (NO)), anti-vascular and anti-angiogenic effects are considered to be major mechanisms of action based on in vitro and animal studies. In cancer patients of Phase I study, DMXAA also exhibited various biological effects, including induction of TNF-alpha, serotonin and NO, which are consistent with those effects observed in in vitro and animal studies. Preclinical studies indicated that DMXAA had more potent anti-tumour activity compared to flavone-8-acetic acid (FAA). In contrast to FAA that did not show anti-tumour activity in cancer patients, DMXAA (22 mg/kg by intravenous infusion over 20 min) resulted in partial response in one patient with metastatic cervical squamous carcinoma in a Phase I study where 65 cancer patients were enrolled in New Zealand. The maximum tolerated dose (MTD) in mouse, rabbit, rat and human was 30, 99, 330, and 99 mg/kg respectively. The dose-limiting toxicity of DMXAA in cancer patients included acute reversible tremor, cognitive impairment, visual disturbance, dyspnoea and anxiety. The plasma protein binding and distribution into blood cells of DMXAA are dependent on species and drug concentration. DMXAA is extensively metabolised, mainly by glucuronidation of its acetic acid side chain and 6-methylhydroxylation, giving rise to DMXAA acyl glucuronide (DMXAA-G), and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), which are excreted into bile and urine. DMXAA-G has been shown to be chemically reactive, undergoing hydrolysis, intramolecular migration and covalent binding. Studies have indicated that DMXAA glucuronidation is catalysed by uridine diphosphate glucuronosyltransferases (UGT1A9 and UGT2B7), and 6-methylhydroxylation by cytochrome P450 (CYP1A2). Non-linear plasma pharmacokinetics of DMXAA has been observed in animals and patients, presumably due to saturation of the elimination process and plasma protein binding. Species differences in DMXAA plasma pharmacokinetics have been observed, with the rabbit having the greatest plasma clearance, followed by the human, rat and mouse. In vivo disposition studies in these species did not provide an explanation for the differences in MTD. Co-administration of DMXAA with other drugs has been shown to result in enhanced anti-tumour activity and alterations in pharmacokinetics, as reported for the combination of DMXAA with melphalan, thalidomide, cyproheptadine, and the bioreductive agent tirapazamine, in mouse models. Species-dependent DMXAA-thalidomide pharmacokinetic interactions have been observed. Co-administration of thalidomide significantly increased the plasma area of the plasma concentration-time curve (AUC) of DMXAA in mice, but had no effect on DMXAA's pharmacokinetics in the rat. It appears that the pharmacological and toxicological properties of DMXAA as a new biological response modifier are unlikely to be predicted based on preclinical studies. Similar to many biological response modifiers, DMXAA alone did not show striking anti-tumour activity in patients. However, preclinical studies of DMXAA-drug combinations indicate that DMXAA may have a potential role in cancer treatment when co-administered with other drugs. Further studies are required to explore the molecular targets of DMXAA and mechanisms for the interactions with other drugs co-administered during combination treatment, which may allow for the optimisation of DMXAA-based chemotherapy.

Strain differences in the liver microsomal metabolism of the experimental anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in mice

The experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is mainly metabolised by acyl glucuronidation and to a lesser degree by 6-methyl hydroxylation. Strain differences in the maximum tolerated dose (MTD) of DMXAA in mice have been observed. The aim of this study was to compare the kinetics of DMXAA acyl glucuronidation and 6-methylhydroxylation in five various mouse strains, and correlate the in vitro metabolism data with MTD observed. In all mouse strains studied, DMXAA acyl glucuronidation and 6-methylhydroxylation in the liver microsomes followed Michaelis–Menten kinetics. Significant strain variations in the kinetic parameters ( K m, V max and K m/ V max, i.e., CL int) for DMXAA acyl glucuronidation and 6-methylhydroxylation in mouse liver microsomes were observed. A 2–6-fold variation was spanned across strains for K m, V max and CL int, respectively, for DMXAA glucuronidation and 6-methylhydroxylation. The rank order for total CL int by glucuronidation and 6-methylhydroxylation was BDF1 (1.70 ml/min per g)>wild type of mice lacking IFN-γ receptor (0.80 ml/min per g)>nude mice (0.70 ml/min per g)>Swiss CD mice (0.56 ml/min per g)>C57Bl/6 mice (0.46 ml/min per g), with a 4-fold variation between the mouse strain of the highest and lowest CL int. There was no significant correlation between total CL int and MTD ( r 2=0.88, P>0.05), but the rank order for CL int was consistent with that for MTD. These results suggested that there were significant strain differences in DMXAA metabolism in mouse liver microsomes and the strain-related differences in the metabolism of DMXAA did not provide an explanation for the strain differences in the MTD.

Gender differences in the metabolism and pharmacokinetics of the experimental anticancer agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

Marked gender differences in the pharmacokinetics of many drugs have been reported. For the investigational anticancer drug, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), negligible gender differences in the plasma pharmacokinetics have been observed in mice. The gender effects on the plasma pharmacokinetics of DMXAA were further investigated using the rat model. In addition, the in vitro metabolism and plasma protein binding of DMXAA in male and female mice, rats and humans were investigated. DMXAA was administered to male and female rats by intravenous injection. DMXAA and its major metabolites formed in liver microsomes were determined by HPLC. Unbound DMXAA in plasma was separated by ultrafiltration followed by HPLC determination. In vivo kinetic studies indicated that female rats had 60%, 55% and 73% higher area under the plasma concentration-time curve (AUC) of DMXAA (2413 +/- 188 vs 1505 +/- 312 microM x h, P<0.05), elimination half-life (2.40 +/- 0.45 vs 1.55 +/- 0.33 h) and maximal plasma concentration (Cmax) (1236 +/- 569 vs 716 +/- 280 micro M), but 61% lower plasma clearance than male rats. In vitro studies indicated that male rats had a 67% higher glucuronidation activity (0.75 +/- 0.03 nmol/min per mg) than female rats (0.45 +/- 0.01 nmol/min per mg), resulting in a 96% faster intrinsic clearance (CL(int)) in the males than the females (6.36 +/- 0.65 vs 3.24 +/- .42 ml/min per g, P< 0.05). In contrast, female rats had 25% higher 6-methylhydroxylation activity (0.045 +/- 0.003 nmol/min per mg) than male rats (0.036 +/- 0.002 nmol/min per mg), resulting in a 57% faster intrinsic clearance (CL(int)) in the females than males (0.36 +/- 0.06 vs 0.23 +/- 0.05 ml/min per g). Overall, total CL(int) by both glucuronidation and 6-methylhydroxylation in male rats was 83% higher than in female rats (6.59 +/- 2.11 vs 3.60 +/- 1.07 nmol/min per g). Men ( n=4) had a significantly lower ( P<0.05) CL(int) for glucuronidation than women ( n=10), but a higher CL(int) for 6-methylhydroxylation, resulting in significantly higher total CL(int) in women than men (5.63 vs 8.33 nmol/min per g). There was no significant difference in either the total plasma protein or albumin concentration between male and female mice, rats or humans. There were significant gender-related differences in the metabolism and pharmacokinetics in the rat, in contrast to the mouse. This indicates a limited usefulness of the rat as a model for the study of DMXAA metabolism in relation to gender differences, although the gender differences in the in vitro metabolic capacity for DMXAA may provide an explanation for the gender differences in the pharmacokinetics in rats. Data from human liver microsomes may allow the prediction of gender effects in the in vivo pharmacokinetics of DMXAA.

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C 18 column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 m M ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μ M in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μ M. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.