Analysis of radiation-induced changes to human melanoma cultures using a mathematical model.

OBJECTIVES: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. MATERIALS AND METHODS: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G(1)-, S- and G(2)/M-phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. RESULTS: The model demonstrated that irradiation at 9 Gy induced G(2)-phase arrest in all lines for at least 96 h. Two cell lines with wild-type p53 status additionally exhibited G(1)-phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G(1)/S and G(2)/M-phase transitions, was broadly comparable with results of clonogenic assays. CONCLUSIONS: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long-term cell cycle arrest.

Cell cycle times of short-term cultures of brain cancers as predictors of survival.

Tumour cytokinetics estimated in vivo as potential doubling times (T(pot) values) have been found to range in a variety of human cancers from 2 days to several weeks and are often related to clinical outcome. We have previously developed a method to estimate culture cycle times of short-term cultures of surgical material for several tumour types and found, surprisingly, that their range was similar to that reported for T(pot) values. As T(pot) is recognised as important prognostic variable in cancer, we wished to determine whether culture cycle times had clinical significance. Brain tumour material obtained at surgery from 70 patients with glioblastoma, medulloblastoma, astrocytoma, oligodendroglioma and metastatic melanoma was cultured for 7 days on 96-well plates, coated with agarose to prevent proliferation of fibroblasts. Culture cycle times were estimated from relative (3)H-thymidine incorporation in the presence and absence of cell division. Patients were divided into two groups on the basis of culture cycle times of < or =10 days and >10 days and patient survival was compared. For patients with brain cancers of all types, median survival for the < or =10-day and >10-day groups were 5.1 and 12.5 months, respectively (P=0.0009). For 42 patients with glioblastoma, the corresponding values were 6.5 and 9.0 months, respectively (P=0.03). Lower grade gliomas had longer median culture cycle times (16 days) than those of medulloblastomas (9.9 days), glioblastomas (9.8 days) or melanomas (6.7 days). We conclude that culture cycle times determined using short-term cultures of surgical material from brain tumours correlate with patient survival. Tumour cells thus appear to preserve important cytokinetic characteristics when transferred to culture.

Pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (AS1404), a novel vascular disrupting agent, in phase I clinical trial.

PURPOSE: 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. METHODS: DMXAA was administered as a 20-min i.v. infusion every 3 weeks and doses were escalated in cohorts of patients according to a predefined schema. PK samples were taken over the first 24 h of at least the first cycle. RESULTS: DMXAA was administered to 63 patients at 19 dose levels from 6 to 4,900 mg m(-2), and 3,700 mg m(-2) was established as the maximum tolerated dose. The PK observed over the dose range showed a non-linear fall in clearance from 16.1 to 1.42 l h(-1) m(-2) and resultant increase in the area under the concentration-time curve (AUC) from 1.29 to 12,400 microM h. In contrast, the increase in peak plasma concentrations from 2.17 to 1,910 microM approximated linearity. DMXAA was highly protein-bound to albumin (>99%) until saturation occurred at higher doses, leading to a rapid increase in the free fraction (up to 20%) and greater concentrations of DMXAA bound to non-albumin proteins. However, the main determinant of the non-linearity of the PK appeared to be sequential saturation of elimination mechanisms, which include hydroxylation, glucuronidation and perhaps hepatic transport proteins. This resulted in an exaggerated non-linear increase in free DMXAA plasma concentrations and AUC compared to total drug. CONCLUSIONS: The PK of DMXAA are well-defined, with a consistent degree of non-linearity across a very large dose range.

In vitro modelling of human tumour behaviour in drug discovery programmes.

Human tumour cell lines have played an important part in our understanding of cancer and have been used extensively in the discovery and characterisation of new chemotherapeutic drugs. A potential weakness of such cell lines is that they may have lost important properties originally possessed in vivo, including potential targets for therapy. This review discusses how possible differences between tumour cells in cancer patients and cell lines might be identified by the use of short-term cultures of human tumour cells taken directly from cancer tissue, termed here primary cultures. Cell-cycle time is one important difference between tumours and cell lines and it is known that the cell-cycle times of primary cultures cover the same wide range as estimated in vivo cell-cycle times. Because tumour cells have at least two pathways to cell death, one from interphase and one from mitosis, changes in cell-cycle length can modify the balance of such pathways. Responses of primary cultures to DNA-damaging drugs and inhibitors of growth factor receptors also differ from those of cell lines, suggesting that the process of developing a cell line can result in the loss of important cellular responses. Without an appreciation of these changes our ability to discover new targets for the development of improved cancer therapy may be jeopardised. The identification of cell lines that preserve potential targets is an important goal in cancer biology and research using primary cultures will help in this identification.

Relationship between tumour endothelial cell apoptosis and tumour blood flow shutdown following treatment with the antivascular agent DMXAA in mice.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is currently undergoing clinical evaluation as an antivascular agent for the treatment of cancer. We have previously demonstrated that DMXAA induces apoptosis of vascular endothelial cells in murine tumour sections and in a breast carcinoma biopsy from one patient in a Phase I trial. We wished to determine the tissue selectivity of this effect and its relationship to induced blood flow changes. Mice with Colon 38 tumours were treated with DMXAA and tissues were examined for apoptosis by TdT-mediated dUTP nick-end labelling (TUNEL). Hoechst 33342 was used to stain functional vessels, with the loss of stained vessels used as a measure of tumour vascular collapse. Treatment with DMXAA at 25 mg kg(-1), its maximum tolerated dose (MTD), showed, after 3 h, a 12-fold increase in TUNEL staining of tumour vascular endothelial cells. In contrast, tissue from the heart, brain, liver and spleen showed no increase. Induction of apoptosis in tumour tissue was both dose-dependent, observable at doses as low as 5 mg kg(-1), and time-dependent. Apoptosis was significantly lower in Colon 38 tumours of mice, with a targeted disruption in the TNF gene (TNF(-/-)), or in the TNF receptor 1 gene (TNFR(-/-)), as compared with that in wild-type mice. Increasing the DMXAA dose to 50 mg kg(-1) in these knockout mice raised tumour apoptosis to a level comparable to that induced in wild-type mice given DMXAA at the MTD. For all the data, a significant correlation (r=0.94; P<0.001) was found between logarithmic percentage apoptosis induction and the logarithmic density of Hoechst-stained vessels. These results suggest that blood flow inhibition caused by DMXAA is tumour tissue-specific and is a consequence of induction of apoptosis in tumour vascular endothelial cells.

Clinical aspects of a phase I trial of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent.

The antitumour action of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is mediated through tumour-selective antivascular effects and cytokine induction. This clinical phase I trial was conducted to examine its toxicity, maximum tolerated dose, pharmacokinetics (PK) and pharmacodynamics (PD). A secondary objective was to assess its antitumour efficacy. DMXAA was administered every 3 weeks as a 20-min i.v. infusion. Dose escalation initially followed a modified Fibonacci schema but was also guided by PK and toxicity. A total of 63 patients received 161 courses of DMXAA over 19 dose levels ranging from 6 to 4900 mg m(-2). DMXAA was well tolerated at lower doses and no drug-related myelosuppression was seen. Rapidly reversible dose-limiting toxicities were observed at 4900 mg m(-2), including confusion, tremor, slurred speech, visual disturbance, anxiety, urinary incontinence and possible left ventricular failure. Transient prolongation of the corrected cardiac QT interval was seen in 13 patients evaluated at doses of 2000 mg m(-2) and above. A patient with metastatic cervical carcinoma achieved an unconfirmed partial response at 1100 mg m(-2), progressing after eight courses. The results of PK and PD studies are reported separately. DMXAA has antitumour activity at well-tolerated doses.

Paclitaxel induces nucleolar enlargement in dorsal root ganglion neurons in vivo reducing oxaliplatin toxicity.

Paclitaxel and oxaliplatin are promising drugs for combination trials but both induce peripheral neurotoxicity. To investigate this toxicity, 10-week-old female Wistar rats were given single intraperitoneal doses of paclitaxel and oxaliplatin, alone or in combination. Neurotoxicity was assessed by L5 dorsal root ganglion morphometry and H-reflex-related sensory nerve conduction velocity. Platinum concentrations in dorsal root ganglia and plasma were measured by inductively coupled plasma mass spectrometry. Dorsal root ganglion nucleolus size was significantly increased following single doses of paclitaxel of 10 and 20 mg kg(-1) at 24 h and 6 days (P<0.02). In contrast, dorsal root ganglion nucleolus size was significantly decreased following single doses of oxaliplatin ranging from 3 to 30 mg kg(-1) at time points ranging from 2 h to 14 days. Sensory nerve conduction velocity was altered after a single dose of oxaliplatin but not after paclitaxel. In combination with oxaliplatin, paclitaxel did not alter the plasma pharmacokinetics or dorsal root ganglion accumulation of oxaliplatin-derived platinum. However, prior paclitaxel inhibited oxaliplatin-induced reductions of dorsal root ganglion nucleolar diameter (P<0.02). Sensory nerve conduction velocity was reduced after oxaliplatin alone (P&<0.05) but unchanged when paclitaxel was given before oxaliplatin. In conclusion, paclitaxel induces nucleolar enlargement in dorsal root ganglion neurons after pharmacologically relevant doses in vivo and reduces oxaliplatin nucleolar damage and neurotoxicity.

NF-kappa B activation in vivo in both host and tumour cells by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer agent developed in this centre, has an antivascular action and causes regression of transplantable murine tumours that is mediated partially by the intratumoral production of tumour necrosis factor (TNF). DMXAA activates the nuclear factor-kappaB (NF-kappaB) transcription factor, which is involved in TNF synthesis and has also been suggested to mediate resistance to TNF. We wished to determine whether tumour cell NF-kappaB activation modulated the in vitro and in vivo effects of DMXAA. We compared the response of the 70Z/3 pre-B lymphoma cell line with that of its mutant 1.3E2 sub-line, which has a defective gamma-subunit of IKK, the kinase that phosphorylates IkappaB leading to NF-kappaB activation. As shown by electrophoretic mobility shift assays (EMSAs), DMXAA induced in vitro translocation of NF-kappaB (p50 and p65 subunits) into the nucleus of 70Z/3 cells, but not of 1.3E2 cells. However, when the cell lines were then grown as subcutaneous tumours in mice and treated with DMXAA (25 mg/kg), activation of NF-kappaB was found in nuclear extracts prepared from both 70/Z3 and 1.3E2 tumours, as well as from Colon 38 tumours that were used for comparison. This suggests that DMXAA induces NF-kappaB responses in host components of the tumour. Tumours grown from both 70Z/3 and 1.3E2 cells were found to regress completely following DMXAA treatment. Thus, the antitumour action of DMXAA appears to be independent of the ability of the target tumour cell population to induce NF-kappaB expression. Moreover, activation of NF-kappaB in the tumour cell did not confer resistance to DMXAA-induced therapy.

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.

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.

Nucleolar damage correlates with neurotoxicity induced by different platinum drugs.

Platinum-based drugs are very useful in cancer therapy but are associated with neurotoxicity in the clinic. To investigate the mechanism of neurotoxicity, dorsal root ganglia of rats treated with various platinum drugs were studied. Cell body, nuclear and nucleolar dimensions of dorsal root ganglia sensory nerve cells were measured to determine morphological toxicity. Sensory nerve conduction velocity was measured to determine functional toxicity. After a single dose of oxaliplatin (10 mg kg(-1)), no significant change in nuclear and cell body diameter was seen but decreased nucleolar size was apparent within a few hours of treatment. Changes in nucleolar size were maximal at 24 hours, recovered very slowly and showed a non-linear dependence on oxaliplatin dose (r(2)= 0.99). Functional toxicity was delayed in onset until 14 days after a single dose of oxaliplatin but eventually recovered 3 months after treatment. Multiple doses of cisplatin, carboplatin, oxaliplatin, R, R-ormaplatin and S, S-ormaplatin were also associated with time-dependent reduction in nucleolar size. A linear correlation was obtained between the rate of change in nucleolar size during multiple dose treatment with the series of platinum drugs and the time taken for the development of altered sensory nerve conduction velocity (r(2)= 0.86;P< 0.024). Damage to the nucleolus of ganglionic sensory neurons is therefore linked to the neurotoxicity of platinum-based drugs, possibly through mechanisms resulting in the inhibition of rRNA synthesis.

Measurement of plasma 5-hydroxyindoleacetic acid as a possible clinical surrogate marker for the action of antivascular agents.

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.

Establishment of primary human meningiomas as subcutaneous xenografts in mice.

Meningiomas are the most frequently occurring benign central nervous system tumours. We determined whether a subcutaneous animal model of meningioma was feasible by implanting fresh meningioma tissue from six patients into 60 athymic (nude) mice, either as tissue blocks (38 mice) or as cell suspensions (22 mice). The tumour take-rates were 74% (block) and 50% (suspension), and the xenografts retained the original tumour grade and subtype morphology by light microscopy. Comparison of cell proliferation markers in xenografts and original tumours gave similar immunohistochemical score rates for Ki-67, but not for PCNA. With the exception of one atypical tumour surgical specimen, all tumours lacked p53 immunopositivity. Transmission electron microscopy of sections of tumour xenografts revealed ultrastructural features, including desmosomes and desmosome-like structures, characteristic of well-differentiated meningiomas. The xenografts grew progressively with a volume increase of more than 10-fold over 6-11 months and an apparent doubling time of 16 weeks. This study demonstrates the utility of the subcutaneous meningioma xenograft as a model for further biological and therapeutic studies.

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma.

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGFbeta1 and TGFbetaRII act as a functional unit in the TGFbeta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGFbetaRII gene, but none in IGFRII or TGFbeta1. An association was found between TGFbetaRII mutations and histology, with 2 out of 3 clear cell carcinomas having TGFbetaRII mutations. This data supports other evidence from mutational analysis of the PTEN and beta-catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes.

The antitumour agent 5,6-dimethylxanthenone-4-acetic acid acts in vitro on human mononuclear cells as a co-stimulator with other inducers of tumour necrosis factor.

5,6-dimethylxanthenone-4-acetic acid (DMXAA), currently in phase I trials, demonstrates excellent activity against transplantable murine tumours with established vasculature. The induction of cytokines, particularly of tumour necrosis factor (TNF), appears to be critical to its action. We investigated TNF induction by DMXAA in cultured human peripheral blood leucocytes (HPBL). TNF was measured by an enzyme-linked immunosorbent assay after 8 h, and NF-kappaB induction by electrophoretic mobility shift assays (EMSA) after 2 h. DMXAA (800 microg/ml) had no effect alone on TNF production but augmented, by up to 4-fold, the ability of bacterial lipopolysaccharide (LPS) to induce TNF. Previously reported results showing TNF production by DMXAA alone were traced to the presence in an earlier batch of DMXAA of a small amount of LPS, the action of which could be blocked by polymyxin B. DMXAA stimulated TNF production by deacylated LPS, which alone had little effect. An antibody (MEM-18) to the CD14 receptor, while blocking the induction of TNF by LPS, enabled DMXAA to both synthesise TNF and induce NF-kappaB. The structurally related drug, flavone acetic acid (FAA), did not induce TNF or synergise with anti-CD14 antibody. DMXAA strongly augmented the ability of suboptimal concentrations of interleukin-1 (IL-1) (25 ng/ml), okadaic acid (OA) (20 ng/ml) and phorbol-12-myristate-13-acetate (PMA) (5 ng/ml) to induce TNF production, suggesting that it affects multiple pathways converging on NF-kappaB activation. Sodium salicylate, a drug reported to inhibit the beta-subunit of IkappaB kinase (IKK), appeared to competitively inhibit TNF production by DMXAA in the presence of anti-CD14 antibody. Taken together, the results indicate DMXAA acts in vitro on HPBL to co-stimulate TNF production by a wide variety of agents, and suggests that IKK is the target that mediates this action.

Identification and reactivity of the major metabolite (beta-1-glucuronide) of the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in humans.

1. The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is extensively metabolized by glucuronidation and 6-methylhydroxylation, resulting in DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA). 2. The major human urinary metabolite of DMXAA was isolated and purified by a solid-phase extraction (SPE) method. The isolated metabolite was hydrolysed to free DMXAA by strong base, and by beta-glucuronidase. Liquid chromatography-mass spectrometry (LC-MS) and spectral data indicated the presence of a molecular ion [M + 1]+ at m/z 459, which was consistent with the molecular weight of protonated DMXAA-G. 3. The glucuronide was unstable in buffer at physiological pH, plasma and blood with species variability in half-life. Hydrolysis and intramolecular migration were major degradation pathways. 4. In vitro and in vivo formation of DMXAA-protein adducts was observed. The formation of DMXAA-protein adducts in cancer patients receiving DMXAA was significantly correlated with plasma DMXAA-G concentration and maximum plasma DMXAA concentration. 5. At least five metabolites of DMXAA were observed in patient urine, with up to 60% of the total dose excreted as DMXAA-G, 5.5% as 6-OH-MXAA and 4.5% as the glucuronide of 6-OH-MXAA. 6. These data suggest that the major metabolite in patients' urine is DMXAA beta-1-glucuronide, which may undergo hydrolysis, molecular rearrangement and covalent binding to plasma protein. The reactive properties of DMXAA-G may have important implications for the pharmacokinetics, pharmacodynamics and toxicity of DMXAA.

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.

Synthesis and cytotoxic activity of 7-oxo-7H-dibenz[f,ij]isoquinoline and 7-oxo-7H-benzo[e]perimidine derivatives.

A series of 7-oxo-7H-dibenz[f,ij]isoquinoline and 7-oxo-7H-benzo[e]perimidines bearing cationic side chains were prepared from aminoanthraquinones. The perimidines were prepared from 1-aminoanthraquinone by initial condensation with urea or dimethylacetamide. A series of 2-, 4-, 8-, and 11-carboxy derivatives of the dibenzisoquinolines were prepared from aminoanthraquinonecarboxylic acids. The cationic derivatives were prepared from these via amide, amine, or methylene linkers to study the effects of side chain positioning on biological activity. Within the series of carboxamide-linked compounds, the order of increasing cytotoxicity was 8- < 4- < 2- < 11-. The 2- and 4-carboxamides showed substantial growth delays against in vivo subcutaneous colon 38 tumors in mice, but the 11-carboxamide had curative activity in this refractory model and is being investigated further.

Extravascular transport of the DNA intercalator and topoisomerase poison N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA): diffusion and metabolism in multicellular layers of tumor cells.

There is considerable evidence that DNA intercalating drugs fail to penetrate tumor tissue efficiently. This study used the multicellular layer (MCL) experimental model, in conjunction with computational modeling, to test the hypothesis that a DNA intercalator in phase II clinical trial, N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA), has favorable extravascular transport properties. Single cell uptake and metabolism of DACA and the related but more basic aminoacridine 9-[3-(dimethylamino)propylamino]acridine (DAPA), and penetration through V79 and EMT6 MCL, were investigated by high-performance liquid chromatography. DACA was accumulated by cells to a lesser extent than DAPA and was metabolized to the previously unreported acridan by V79 but not EMT6 cells. Despite this metabolism, flux of DACA through MCL was much faster than that of DAPA. Modeling MCL transport as diffusion with reaction (metabolism and reversible binding) showed that the faster flux of DACA was due to a 3-fold higher free drug diffusion coefficient and 10-fold lower binding site density. The MCL transport parameters were used to develop a spatially resolved pharmacokinetic model for the extravascular compartment in tumors, which provided a reasonable prediction of measured average tumor concentrations from plasma pharmacokinetics in mice. Area under the curve was essentially independent of distance from blood vessels, although the combined pharmacokinetic/pharmacodynamic model predicted a modest decrease in cytotoxicity (from 1.8 to 1.1 logs of cell kill) across a 125-microm region. In conclusion, this study demonstrates that it is possible to design DNA intercalators that diffuse efficiently in tumor tissue, and that there is little impediment to DACA transport over distances required for its antitumor action.

Antitumor activity of XR5944, a novel and potent topoisomerase poison.

Inhibitors of topoisomerases are widely used in the treatment of cancer, including inhibitors of topoisomerase I (camptothecin analogs such as irinotecan and topotecan) and topoisomerase II (etoposide and doxorubicin). The novel bis-phenazine, XR5944, is a joint inhibitor of topoisomerase I and II as shown by the stabilization of topoisomerase-dependent cleavable complexes. XR5944 demonstrated exceptional activity against human and murine tumor cells in vitro and in vivo. In a range of cell lines XR5944 (IC50 0.04-0.4 nM) was significantly more potent than TAS-103, originally proposed as a joint topoisomerase I and II inhibitor, as well as agents specific for topoisomerase I or II (topotecan, doxorubicin and etoposide). In addition, XR5944 was unaffected by atypical drug resistance and retained significant activity in cells overexpressing P-glycoprotein or multidrug resistance-associated protein. Antitumor efficacy of XR5944 was demonstrated in human carcinoma xenograft models (H69 small cell lung cancer and HT29 colon). In the HT29 model, which is relatively unresponsive to chemotherapy, XR5944 (15 mg/kg i.v., q4dx3) induced tumor regression in the majority of animals (six of eight), whereas TAS-103, dosed at its maximum tolerated dose (45 mg/kg i.v., q7dx3), only induced a delay in tumor growth compared with control animals. In the H69 model, low doses of XR5944 (5 mg/kg i.v., qdx5/week for 2 weeks or 10-15 mg/kg i.v., q4dx3), induced complete tumor regression in the majority of animals. In contrast, topotecan (20 mg/kg i.v., q4dx3) or etoposide (30 mg/kg i.v., q5dx5) only slowed the tumor growth rate. These studies show that XR5944 is a highly active novel anticancer agent that is well tolerated at efficacious doses.