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Magnetic resonance imaging (MRI) can measure the effects of therapies targeting the tumor vasculature and has demonstrated that vascular-damaging agents (VDA) induce acute vascular shutdown in tumors in human and animal models. However, at subtherapeutic doses, blood flow may recover before the induction of significant levels of necrosis. We present the relationship between changes in MRI biomarkers and tumor necrosis. Multiple MRI measurements were taken at 4.7 T in athymic rats (n = 24) bearing 1.94 ± 0.2-cm3 subcutaneous Hras5 tumors (ATCC 41000) before and 24 hours after clinically relevant doses of the VDA, ZD6126 (0-10 mg/kg, i.v.). We measured effective transverse relaxation rate (R2*), initial area under the gadolinium concentration-time curve (IAUGC60/150), equivalent enhancing fractions (EHF60/150), time constant (Ktrans), proportion of hypoperfused voxels as estimated from fit failures in Ktrans analysis, and signal intensity (SI) in T2-weighted MRI (T2W). ZD6126 treatment induced < 90% dose-dependent tumor necrosis at 10 mg/kg; correspondingly, SI changes were evident from T2W MRI. Although R2* did not correlate, other MRI biomarkers significantly correlated with necrosis at doses of ≥ 5 mg/kg ZD6126. These data on Hras5 tumors suggest that the quantification of hypoperfused voxels might provide a useful biomarker of tumor necrosis.

The response of radiation-induced fibrosarcoma1 (RIF-1) tumors treated with the vascular-disrupting agent (VDA) ZD6126 was assessed by in vivo and ex vivo1H magnetic resonance spectroscopy (MRS) methods. Tumors treated with 200 mg/kg ZD6126 showed a significant reduction in total choline (tCho) in vivo 24 hours after treatment, whereas control tumors showed a significant increase in tCho. This response was investigated further within both ex vivo unprocessed tumor tissues and tumor tissue metabolite extracts. Ex vivo high-resolution magic angle spinning (HRMAS) and 1H MRS of metabolite extracts revealed a significant reduction in phosphocholine and glycerophosphocholine in biopsies of ZD6126-treated tumors, confirming in vivo tCho response. ZD6126-induced reduction in choline compounds is consistent with a reduction in cell membrane turnover associated with necrosis and cell death following disruption of the tumor vasculature. In vivo tumor tissue water diffusion and lactate measurements showed no significant changes in response to ZD6126. Spin-spin relaxation times (T2) of water and metabolites also remained unchanged. Noninvasive 1H MRS measurement of tCho in vivo provides a potential biomarker of tumor response to VDAs in RIF-1 tumors.

The effective magnetic resonance imaging (MRI) transverse relaxation rate R2* was investigated as an early acute marker of the response of rat GH3 prolactinomas to the vascular-targeting agent, ZD6126. Multigradient echo (MGRE) MRI was used to quantify R2*, which is sensitive to tissue deoxyhemoglobin levels. Tumor R2* was measured prior to, and either immediately for up to 35 minutes, or 24 hours following administration of 50 mg/kg ZD6126. Following MRI, tumor perfusion was assessed by Hoechst 33342 uptake. Tumor R2* significantly increased to 116 ± 4% of baseline 35 minutes after challenge, consistent with an ischemic insult induced by vascular collapse. A strong positive correlation between baseline R2* and the subsequent increase in R2* measured 35 minutes after treatment was obtained, suggesting that the baseline R2* is prognostic for the subsequent tumor response to ZD6126. In contrast, a significant decrease in tumor R2* was found 24 hours after administration of ZD6126. Both the 35-minute and 24-hour R2* responses to ZD6126 were associated with a decrease in Hoechst 33342 uptake. Interpretation of the R2* response is complex, yet changes in tumor R2* may provide a convenient and early MRI biomarker for detecting the antitumor activity of vascular-targeting agents.

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

Methods of monitoring drug toxicity in off-target organs are very important in the development of effective and safe drugs. Standard 2-D techniques, such as histology, are prone to sampling errors and can miss important information. We demonstrate a novel application of optical computed tomography (CT) imaging to quantitatively assess, in 3-D, the response of adult murine spleen to off-target drug toxicity induced by treatment with the vascular disrupting agent ZD6126. Reconstructed images from optical CT scans sensitive to haemoglobin absorption reveal detailed, high-contrast 3-D maps of splenic structure and microvasculature. A significant difference in total splenic volume was found between vehicle and ZD6126-treated cohorts, with mean volumes of 61 ± 3 mm 3 and 44 ± 3 mm 3 respectively (both n = 3, p = 0.05). Textural statistics for each sample were calculated using grey-level co-occurrence matrices (GLCMs). Standard 2-D GLCM analysis was found to be slice-dependent while 3-D GLCM contrast and homogeneity analysis resulted in separation of the vehicle and ZD6126-treated cohorts over a range of length scales. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the influence of two types of vascular damaging agents (VDAs) (DMXAA vs. ZD6126) and sequence of administration (VDA 24 h before HYP vs. HYP 1 h before VDA) to evaluate the effect on hypericin (HYP) accumulation and distribution in necrotic tumors. Methods: Frozen sections of dorsally inoculated RIF-1 tumors were analyzed by fluorescence microscopy and H&E stained for histological evaluation. The localization of HYP was assessed both qualitatively and semi-quantitatively in necrotic tumor, viable tumor, or nontarget host tissue. Results: Whereas the type of VDA did not influence HYP accumulation and distribution, a clear advantage could be seen when administering VDA 24 h before HYP compared to HYP 1 h before VDA, pointing toward the absence of a 'trapping' mechanism. In DMXAA-treated and not in ZD6126-treated tumors, spotty fluorescence was observed which is likely to be a consequence of neutrophil phagocytosis. Dexamethasone treatment neither did influence this phenomenon nor did change HYP uptake in necrotic tumor. Conclusions: We conclude that HYP accumulation is optimal when it is administered after VDA injection. We also found that HYP accumulation in necrosis is not changed when using VDAs with different working mechanisms. This insight provides a rationale for tumor necrosis therapy (TNT) using iodine-131-labeled hypericin ([I]-HYP) in combination with VDAs. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the use of the transverse magnetic resonance imaging (MRI) relaxation rate R2 ∗ (s-1) as a biomarker of tumor vascular response to monitor vascular disrupting agent (VDA) therapy. Methods and Materials: Multigradient echo MRI was used to quantify R2 ∗ in rat GH3 prolactinomas. R2∗ is a sensitive index of deoxyhemoglobin in the blood and can therefore be used to give an index of tissue oxygenation. Tumor R2 ∗ was measured before and up to 35 min after treatment, and 24 h after treatment with either 350 mg/kg 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or 100 mg/kg combretastatin-A4-phosphate (CA4P). After acquisition of the MRI data, functional tumor blood vessels remaining after VDA treatment were quantified using fluorescence microscopy of the perfusion marker Hoechst 33342. Results: DMXAA induced a transient, significant (p < 0.05) increase in tumor R2 ∗ 7 min after treatment, whereas CA4P induced no significant changes in tumor R2 ∗ over the first 35 min. Twenty-four hours after treatment, some DMXAA-treated tumors demonstrated a decrease in R2 ∗, but overall, reduction in R2 ∗ was not significant for this cohort. Tumors treated with CA4P showed a significant (p < 0.05) reduction in R2 ∗ 24 h after treatment. The degree of Hoechst 33342 uptake was associated with the degree of R2 ∗ reduction at 24 h for both agents. Conclusions: The reduction in tumor R2 ∗ or deoxyhemoglobin levels 24 h after VDA treatment was a result of reduced blood volume caused by prolonged vascular collapse. Our results suggest that DMXAA was less effective than CA4P in this rat tumor model. [Copyright &y& Elsevier]

Purpose: To assess tumor fractional blood volume (ξ), determined in vivo by susceptibility contrast magnetic resonance imaging (MRI) as a noninvasive imaging biomarker of tumor response to the vascular disrupting agent ZD6126. Methods and Materials: The transverse MRI relaxation rate R2 ∗ of rat GH3 prolactinomas was quantified prior to and following injection of 2.5 mgFe/kg feruglose, an ultrasmall superparamagnetic iron oxide intravascular contrast agent, and ξ (%) was determined from the change in R2 ∗. The rats were then treated with either saline or 50 mg/kg ZD6126, and ξ measured again 24 hours later. Following posttreatment MRI, Hoechst 33342 (15 mg/kg) was administered to the rats and histological correlates from composite images of tumor perfusion and necrosis sought. Results: Irrespective of treatment, tumor volume significantly increased over 24 hours. Saline-treated tumors showed no statistically significant change in ξ, whereas a significant (p = 0.002) 70% reduction in ξ of the ZD6126-treated cohort was determined. Hoechst 33342 uptake was associated with viable tumor tissue and was significantly (p = 0.004) reduced and restricted to the rim of the ZD6126-treated tumors. A significant positive correlation between posttreatment ξ and Hoechst 33342 uptake was obtained (r = 0.83, p = 0.002), providing validation of the MRI-derived measurements of fractional tumor blood volume. Conclusions: These data clearly highlight the potential of susceptibility contrast MRI with ultrasmall superparamagnetic iron oxide contrast agents to provide quantitative imaging biomarkers of fractional tumor blood volume at high spatial resolution to assess tumor vascular status and response to vascular disrupting agents. [Copyright &y& Elsevier]

The clinical success of small-molecule vascular disrupting agents (VDAs) depends on their combination with conventional therapies. Scheduling and sequencing remain key issues in the design of VDA–chemotherapy combination treatments. This study examined the antitumour activity of ZD6126, a microtubule destabilising VDA, in combination with paclitaxel (PTX), a microtubule-stabilising cytotoxic drug, and the influence of schedule and sequence on the efficacy of the combination. Nude mice bearing MDA-MB-435 xenografts received weekly cycles of ZD6126 (200 mg kg−1 i.p.) administered at different times before or after PTX (10, 20, and 40 mg kg−1 i.v.). ZD6126 given 2 or 24 h after PTX showed no significant benefit, a result that was attributed to a protective effect of PTX against ZD6126-induced vascular damage and tumour necrosis, a hallmark of VDA activity. Paclitaxel counteracting activity was reduced by distancing drug administrations, and ZD6126 given 72 h after PTX potentiated the VDA's antitumour activity. Schedules with ZD6126 given before PTX improved therapeutic activity, which was paralleled by a VDA-induced increase in cell proliferation in the viable tumour tissue. Paclitaxel given 72 h after ZD6126 yielded the best response (50% tumours regressing). A single treatment with ZD6126 followed by weekly administration of PTX was sufficient to achieve a similar response (57% remissions). These findings show that schedule, sequence and timing are crucial in determining the antitumour efficacy of PTX in combination with ZD6126. Induction of tumour necrosis and increased proliferation in the remaining viable tumour tissue could be exploited as readouts to optimise schedules and maximise therapeutic efficacy.British Journal of Cancer (2007) 97, 888–894. doi:10.1038/sj.bjc.6603969 www.bjcancer.com Published online 11 September 2007 [ABSTRACT FROM AUTHOR]

The in vitro metabolism of M445526 (ZD6126 phenol) was investigated by incubating [14C]-M445526 at a concentration of 10 µg ml-1 with human hepatic microsomes (4 mg ml-1) or human hepatocytes (2 × 106 cells ml-1) for up to 180 min. Following incubation with microsomes and hepatocytes, up to 78% and 40% of [14C]-M445526 was metabolized after 180 and 120 min, respectively. High-performance liquid chromatography (HPLC) with radiochemical detection confirmed extensive metabolism of [14C]-M445526 by microsomes and hepatocytes. Mass spectrometry and 1H-NMR spectroscopy enabled structural identification of up to eight metabolites. Human liver microsomes formed one major (O-desmethyl) and three minor (a further O-desmethyl and two different hydroxylated) phase I metabolites. Human hepatocytes produced one major metabolite, a sulphate conjugate of the major O-desmethyl metabolite formed by microsomes. Four minor metabolites were also formed, primarily by O-demethylation with subsequent glucuronidation. Taken collectively, [14C]-M445526 underwent extensive in vitro metabolism by human liver fractions. These data were confirmed by subsequent human in vivo studies. [ABSTRACT FROM AUTHOR]

Abstract: The subject of this study was the determination of the major urinary and biliary metabolites of [14C]-ZD6126 following i.v. administration to female and male bile duct cannulated rats at 10mg/kg and 20mg/kg, respectively, and male bile duct cannulated dogs at 6mg/kg by HPLC–NMR spectroscopy. ZD6126 is a phosphorylated pro-drug, which is rapidly hydrolysed to the active metabolite, ZD6126 phenol. The results presented here demonstrate that [14C]-ZD6126 phenol is subsequently metabolised extensively by male dogs and both, male and female rats. Recovery of the dose in bile and urine was determined utilising the radiolabel, revealing biliary excretion as the major route of excretion (93%) in dog, with the majority of the radioactivity recovered in both biofluids in the first 6h. In the rat, greater than 92% recovery was obtained within the first 24h. The major route of excretion was via the bile 51–93% within the first 12h. The administered phosphorylated pro-drug was not observed in any of the excreta samples. Metabolite profiles of bile and urine samples were determined by high performance liquid chromatography with radiochemical detection (HPLC–RAD), which revealed a number of radiolabelled components in each of the biofluids. The individual metabolites were subsequently identified by HPLC–NMR spectroscopy and HPLC–MS. In the male dog, the major component in urine and bile was the [14C]-ZD6126 phenol glucuronide, which accounted for 3% and 77% of the dose, respectively. [14C]-ZD6126 phenol was observed in urine at 1% of dose, but was not observed in bile. A sulphate conjugate of demethylated [14C]-ZD6126 phenol was identified in bile by HPLC–NMR and confirmed by HPLC–MS. In the rat, the bile contained two major radiolabelled components. One was identified as the [14C]-ZD6126 phenol glucuronide, the other as a glucuronide conjugate of demethylated [14C]-ZD6126 phenol. However, a marked difference in the proportions of these two components was observed between male and female rats, either due to a sex difference in metabolism or a difference in dose level. The glucuronide conjugate of the demethylated [14C]-ZD6126 phenol was present at higher concentration in the bile of male rats (4–34%), while the phenol glucuronide was present at higher concentration in the bile of female rats (8–70%) over a 0–6h collection period. A third component was only observed in the bile samples (0–6h and 6–12h) of male rats. This was identified as being the same sulphate conjugate of demethylated [14C]-ZD6126 phenol as the one observed in dog bile. The rat urines contained two main metabolites in greatly varying concentrations, namely the demethylated [14C]-ZD6126 phenol glucuronide and the glucuronide of [14C]-ZD6126 phenol. Again, the differences in relative amounts between male and female rats were observed, the major metabolite in the urines from male rats being the demethylated [14C]-ZD6126 phenol (0–17% in 0–24h), whilst the phenol glucuronide, accounting for 0.5–50% of the dose over 0–24h, was the major metabolite in females. Methanolic extracts of the pooled biofluid samples were submitted for HPLC–NMR for the quick identification of the major metabolites. Following a single injection of the equivalent of 6–28ml of the biofluids directly onto the HPLC-column with minimal sample preparation, the metabolites could be largely successfully isolated. Despite severe column overloading, the major metabolites of [14C]-ZD6126 could be positively identified, and the results are presented in this paper. [Copyright &y& Elsevier]

Current experimental and clinical knowledge supports the optimisation of endothelial cell targeting using a strategy combining anti-EGFR drugs with antivascular agents. The purpose of the present study was to examine the effects of the association of ZD6126, an antivascular microtubule-destabilising agent, with gefitinib and irradiation on the growth of six head and neck human cancer cell lines xenografted in nude mice and to study predictive and molecular factors responsible for antitumour effects. CAL33- and Hep-2-grafted cell lines were the most sensitive to ZD6126 treatment, with VEGF levels significantly higher (P=0.0336) in these tumour xenografts compared to Detroit 562- and CAL27-grafted cell lines with relatively low VEGF levels that were not sensitive to ZD6126. In contrast, neither IL8 levels nor EGFR expression was linked to the antitumour effects of ZD6126. ZD6126 in combination with gefitinib resulted in a synergistic cytotoxic interaction with greater antitumour effects than gefitinib alone. The synergistic interaction between ZD6126 and gefitinib was corroborated by a significant decrease in CD31 labelling. The present study may serve for future innovative clinical applications, as it suggests that VEGF tumour levels are possible predictors for ZD6126 antitumour efficacy. It also supports the notion of antitumour supra-additivity when combining gefitinib and ZD6126, and identifies neoangiogenesis as the main determinant of this synergistic combination. [ABSTRACT FROM AUTHOR]

Purpose: To examine the antivascular and antitumor activity of the vascular targeting agent ZD6126 in combination with radiation in lung and head-and-neck (H and N) cancer models. The overall hypothesis was that simultaneous targeting of tumor cells (radiation) and tumor vasculature (ZD6126) might enhance tumor cell killing.Methods and Materials: A series of in vitro studies using human umbilical vein endothelial cells (HUVEC) and in vivo studies in athymic mice bearing human lung (H226) and H and N (squamous cell carcinoma [SCC]1, SCC6) tumor xenografts treated with ZD6126 and/or radiation were performed.Results: ZD6126 inhibited the capillary-like network formation in HUVEC. Treatment of HUVEC with ZD6126 resulted in cell cycle arrest in G2/M, with decrease of cells in S phase and proliferation inhibition in a dose-dependent manner. ZD6126 augmented the cell-killing effect of radiation and radiation-induced apoptosis in HUVEC. The combination of ZD6126 and radiation further decreased tumor vascularization in an in vivo Matrigel angiogenesis assay. In tumor xenografts, ZD6126 enhanced the antitumor activity of radiation, resulting in tumor growth delay.Conclusions: These preclinical studies suggest that ZD6126 can augment the radiation response of proliferating endothelial H and N and lung cancer cells. These results complement recent reports suggesting the potential value of combining radiation with vascular targeting/antiangiogenic agents. [ABSTRACT FROM AUTHOR]

Abstract: Tumours derived from DLD-1 colon adenocarcinoma cells, transfected to either overexpress inducible nitric oxide synthase (clone iNOS-19) or with empty vector (pBAN2R), were utilised to test the hypothesis that tumour expression of iNOS (a) increases tumour angiogenesis and (b) modulates the anti-tumour activity of the vascular disrupting agent ZD6126. Overexpression of iNOS by clone iNOS-19 cells and murine xenografts was confirmed by the Griess assay and western blot analysis respectively. Clone iNOS-19 tumours grew more rapidly than pBAN2R tumours. Tumour perfusion, assessed by Hoechst 33342 uptake, was significantly greater in the clone iNOS-19 tumours (P < 0.001). A significant reduction in the perfusion of only the pBAN2R tumours, compared with control, was obtained 24 h after treatment with an intermediate dose of 100 mg/kg ZD6126 (P < 0.001), whereas 200 mg/kg significantly reduced the perfusion of both tumour types (P < 0.001). Whilst pBAN2R tumour necrosis increased in a dose-dependent manner, significant at 100 and 200 mg/kg ZD6126 (P < 0.05), intermediate doses did not induce a similar degree of necrosis in clone iNOS-19 tumours. A significant reduction in splenic perfusion was found 24 h after treatment with 100 mg/kg ZD6126, primarily associated with the red pulp. Overexpression of iNOS increases tumour growth, the degree of functionally perfused vasculature and angiogenesis, and also confers resistance to the vascular disrupting agent ZD6126. [Copyright &y& Elsevier]

Abstract: Aims: To provide a comprehensive overview of the current state of development of a novel class of anti-cancer drugs, the vascular disrupting agents (VDAs), previously known as vascular targeting agents (VTAs). Materials and methods: A comprehensive review, analysis and commentary of the published medical literature on VDAs was performed. Results: Tumour vascular targeting therapy exploits known differences between normal and tumour blood vessels. VDAs target the pre-existing vessels of tumours (cf anti-angiogenics), and cause vascular shutdown leading to tumour cell death and rapid haemorrhagic necrosis within hours. It is becoming clear that VDAs have overlapping activity with anti-angiogenic drugs, which prevent the formation of new blood vessels. There are two types of VDA. First, biological or ligand-directed VDAs use antibodies, peptides or growth factors to target toxins or pro-coagulants to the tumour endothelium. In contrast, small molecule VDAs work either as tubulin-binding agents or through induction of local cytokine production. VDAs can kill tumour cells resistant to conventional chemotherapy and radiotherapy, and work best on cells in the poorly perfused hypoxic core of tumours, leaving a viable rim of well-perfused tumour tissue at the periphery, which rapidly regrows. Consequently, responses of tumours to VDAs given as single agents have been poor; however, combination therapy with cytotoxic chemotherapy, external-beam radiotherapy, and radioimmunotherapy, which target the peripheral tumour cells, has produced some excellent responses in animal tumours. VDAs are generally well tolerated with different side-effect profiles to current oncological therapies. Dynamic magnetic resonance imaging is most frequently used to obtain a pharmacodynamic end point to determine whether the VDA is acting on its intended target. Conclusions: VDAs are a promising new class of drug, which offer the attractive possibility of inducing responses in all tumour types with combination therapy. [Copyright &y& Elsevier]

Purpose: To evaluate the antitumor efficacy of a vascular targeting strategy that combines an agent that disrupts established tumor blood vessels (ZD6126) with one that interferes with new vessel formation (ZD6474) in models of human renal cell carcinoma (Caki-1) and Kaposi's sarcoma (KSY-1).Methods and Materials: Caki-1 and KSY-1 xenograft-bearing nude mice were treated with ZD6126 and ZD6474 either as single agents or in combination when the tumors reached a size of approximately 200 mm(3). ZD6126 therapy consisted of three doses of 100 mg/kg administered 1, 3, and 5 days after the tumor reached the starting size. ZD6474 was administered daily (25 mg/kg) on Days 1-5. In the combination studies, ZD6474 treatment began immediately after the first dose of ZD6126. The tumor response to treatment was evaluated using a regrowth delay endpoint.Results: Significant tumor growth delays were observed in both tumor models with either agent with the treatment regimen used. In the Caki-1 and KSY-1 models, respectively, ZD6126 treatment resulted in a tumor growth delay of 23 and 26 days and ZD6474 produced a tumor growth delay of 24.5 and 14.5 days. When ZD6126 and ZD6474 were combined, the tumor growth delays increased to 55 (Caki-1) and 86 (KSY-1) days. In the KSY-1 model, the combination therapy also resulted in 3 of 8 long-term tumor-free survivors.Conclusion: These results indicate that statistically significant antitumor efficacy can be achieved using a treatment strategy that combines a therapy that targets the established tumor blood vessels with one that interferes with the process of angiogenesis. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the antitumor efficacy of the vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) in the rodent squamous cell carcinoma (SCC) VII carcinoma model, in combination with boron neutron capture therapy (BNCT).Sodium borocaptate-10B (BSH, 125 mg/kg, i.p.) or l-p-boronophenylalanine-10B (BPA, 250 mg/kg, i.p.) was injected into SCC VII tumor–bearing mice, and 15 min later, ZD6126 (100 mg/kg, i.p.) was administered. Then, the 10B concentrations in tumors and normal tissues were measured by prompt γ-ray spectrometry. On the other hand, for the thermal neutron beam exposure experiment, SCC VII tumor–bearing mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, followed by treatment with a 10B-carrier and ZD6126 in the same manner as the above-mentioned 10B pharmacokinetics analyses. To obtain almost similar intratumor 10B concentrations during neutron exposure, thermal neutron beam irradiation was started from the time point of 30 min after injection of BSH only, 90 min after BSH injection for combination with ZD6126, 120 min after the injection of BPA only, and 180 min after BPA injection for combination with ZD6126. Right after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The clonogenic cell survival assay was also performed in mice given no BrdU.Pharmacokinetics analyses showed that combination with ZD6126 greatly increased the 10B concentrations in tumors after 60 min after BSH injection and after 120 min after BPA injection. The concentrations of 10B from BSH in normal tissues were also raised by combination with ZD6126, although not so clearly as those in tumors. Combination with ZD6126 had almost no effect on the concentrations of 10B from BPA in normal tissues. The clonogenic surviving fractions of total tumor cells and the MN frequencies of both total and Q tumor cells were reduced and increased by combination with ZD6126, respectively, whether BSH or BPA was employed. However, the degrees of these changes in the clonogenic surviving fractions and the MN frequencies were more obviously observed in tumors from BSH-injected mice than from BPA-injected mice, and in Q tumor cells than in total tumor cells regardless of the employed 10B-carrier.Combination with ZD6126 was regarded as more promising in BSH-BNCT than BPA-BNCT, and more effective for enhancing the sensitivity of the Q tumor cells than that of the total tumor cells. This resulted in the decrease in the extended difference in the sensitivity between the total and Q tumor cells caused by the use of 10B-carrier for BNCT. [Copyright &y& Elsevier]

Metastases require a functional blood supply for progressive growth. Thus, therapies that target metastatic vasculature have potential clinical utility. The effects of the vascular-targeting agent (VTA), ZD6126, and the anti-angiogenic agent, ZD6474, on vascular development and function within metastases were compared in an experimental liver metastasis model. Ras-transformed PAP2 fibroblasts were injected into the mesenteric veins of SCID mice to produce a control liver metastasis burden of ∼40% at 14 days. Mice given a single dose of ZD6126 (200 mg/kg, i.p.) on day 13 were examined 24 h later. Histology revealed a significant reduction in metastatic burden, associated with extensive tumor necrosis, increased tumor cell apoptosis and a reduction in tumor-associated vasculature. In vivo videomicroscopy (IVVM) revealed disrupted, non-functional vascular channels within metastases, with no blood flow. Mice given ZD6474 on days 4 to 10 (50 mg/kg daily, oral gavage) were examined on day 11. Histology revealed a lower metastatic burden, significant reductions in metastasis size and vasculature, and a significant increase in tumor cell apoptosis. IVVM revealed extensive reductions in vascularity and blood flow within metastases. Neither ZD6126 nor ZD6474 treatment affected surrounding normal liver tissue. This study shows that both agents can reduce experimental liver metastasis with no apparent effect on normal vasculature. However, these reductions were attained through distinct effects on the metastatic vasculature. Understanding differences in the modes of action of VTAs and anti-angiogenic agents will be important in optimizing their clinical application and in developing appropriate combination strategies. [ABSTRACT FROM AUTHOR]

: PurposeThe aim of this study was to investigate the pathophysiologic effects induced by the novel vascular targeting agent ZD6126 in a C3H mouse mammary carcinoma and to evaluate the agent''s ability to inhibit tumor growth either when given alone or in combination with radiation.: Methods and materialsA C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm3 in size. ZD6126 was dissolved in saline and injected intraperitoneally. Blood perfusion was measured using the RbCl extraction procedure, tumor oxygen (pO2) status was assessed with the Eppendorf electrode, and tumor necrosis was estimated from histologic sections. Radiation (240-kV X-rays) was locally administered to tumors of restrained nonanesthetized mice, and response was assessed using a tumor growth assay.: ResultsZD6126 induced a significant dose- and time-dependent decrease in tumor perfusion, reaching a maximal 70% reduction around 3 h after injecting 150–300 mg/kg. However, full recovery was seen within 6 h. A 200 mg/kg dose significantly decreased tumor oxygenation status at 3 h (median pO2 decreased from 7 to 3 mm Hg; % pO2 values ≤2.5 mm Hg increased from 30% to 55%) and by 24 h had significantly increased necrotic fraction from 14.5% to 25.2%. This ZD6126 dose resulted in a small, yet significant, 1.4 days inhibition of tumor growth when given alone. It also enhanced the tumor response to radiation, giving rise to a significant 1.3-fold increase in the slope of the radiation dose–response curve. Of the normal tissues, only muscle (at 3 h) and spleen (at 6 h) showed any significant reduction in perfusion after injecting 200 mg/kg, but these transient decreases were only 32% and 49%, respectively.: ConclusionsOur preclinical studies clearly demonstrate a tumor-specific reduction in blood perfusion by ZD6126. Although these changes were transient, they were sufficient to increase tumor necrosis, inhibit tumor growth, and enhance radiation response. [Copyright &y& Elsevier]

Advances in the understanding of tumour biology have led to the discovery of new targets that control specific mechanisms essential for tumour spread, growth and survival. In order to fully explore the anticancer potential of these novel approaches, AstraZeneca is developing a broad pipeline of agents targeting a variety of key processes in tumour progression and metastasis. These include two novel antiangiogenic agents, ZD6474 and AZD2171, which are both orally available inhibitors of vascular endothelial growth factor receptor-tyrosine kinase, AZD2171 being a highly potent inhibitor; ZD6474 also has activity against epidermal growth factor receptor tyrosine kinase. Once-daily administration of these agents has been shown to result in effective inhibition of tumour growth in a broad spectrum of human xenograft models. In contrast to this approach, which prevents new vessel formation, the vascular-targeting agent ZD6126 disrupts the microtubular network responsible for maintaining the shape of immature endothelial cells, thereby selectively destroying the existing tumour vasculature and leading to extensive central necrosis. Other agents with a variety of novel antitumour strategies are also in development. These include AZD0530, an orally available Src kinase inhibitor, and AZD3409, an oral prenyl transferase inhibitor, both of which have potential for broad antitumour activity. In addition, an oral, selective, cyclin-dependent kinase inhibitor (AZD5438). [Copyright &y& Elsevier]

Purpose: The present report reviews the preclinical data on combined chemotherapy/vascular targeting agent treatments. Basic principles are illustrated in studies evaluating the antitumor efficacy of the vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) when combined with the anticancer drug cisplatin in experimental rodent (KHT sarcoma) and human renal (Caki-1) tumor models.Methods and Materials: C3H/HeJ and NCR/nu-nu mice bearing i.m. tumors were injected i.p. with ZD6126 (0–150 mg/kg) or cisplatin (0–20 mg/kg) either alone or in combination. Tumor response to treatment was assessed by clonogenic cell survival.Results: Treatment with ZD6126 was found to damage existing neovasculature, leading to a rapid vascular shutdown. Histologic evaluation showed dose-dependent morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. ZD6126 doses that led to pathophysiologic effects also enhanced the tumor cell killing of cisplatin when administered either 24 h before or 1–24 h after chemotherapy. In both tumor models, the administration of a 150 mg/kg dose of ZD6126 1 h after a range of doses of cisplatin resulted in an increase in tumor cell kill 10–500-fold greater than that seen with chemotherapy alone. In contrast, the inclusion of the antivascular agent did not increase bone marrow stem cell toxicity associated with this anticancer drug.Conclusion: The results obtained in the KHT and Caki-1 tumor models indicate that ZD6126 effectively enhanced the antitumor effects of cisplatin therapy. These findings are representative of the marked enhancements generally observed when vascular targeting agents are combined with chemotherapy in solid tumor therapy. [Copyright &y& Elsevier]

Purpose: The aim of these studies was to evaluate factors that contribute to the selectivity of the novel vascular targeting agent ZD6126.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with ZD6126 phenol, and effects on morphology, detachment, and cytotoxicity (sulforhodamine-B dye incorporation) were determined. Hras5-transformed mouse 3T3 fibroblasts were implanted s.c. in athymic nude rats, and effects on the tumor were assessed after either i.v. bolus or 24-h minipump infusion of ZD6126.Results: In vitro, ZD6126 phenol (∼0.1 μm) rapidly (<40 min) destabilized the tubulin cytoskeleton of proliferating endothelial cells, resulting in cell shape change (“rounding up”) and cell detachment at noncytotoxic drug concentrations. In vivo, in rats, an i.v. bolus dose of ZD6126 (20 mg/kg) was rapidly broken down to ZD6126 phenol, which has a short plasma elimination half-life (∼1 h). Peak plasma levels of ZD6126 phenol were well above the level required to induce HUVEC morphology changes in vitro, but cytotoxic concentrations were not maintained. A single i.v. bolus dose (50 and 20 mg/kg) of ZD6126 was well tolerated and resulted in extensive central tumor necrosis in the Hras5 model. Administration of ZD6126 using a 24-h s.c. minipump resulted in decreased (∼30-fold) peak plasma levels, but maintained cytotoxic drug levels over 24 h. Infusion of 50 mg/kg ZD6126 over 24 h was not tolerated. Infusion of 20 mg/kg ZD6126 resulted in increased toxicity compared with the i.v. bolus doses of ZD6126 and did not result in any increased tumor necrosis after 24 h.Conclusion: ZD6126 phenol induces rapid morphologic changes in HUVECs at noncytotoxic drug levels. These rapid morphologic effects combined with the rapid elimination of ZD6126 phenol contribute to the selective effects of ZD6126 on tumor vasculature at well-tolerated doses. [Copyright &y& Elsevier]

Purpose: The aim of this study was to evaluate the antitumor efficacy of the novel vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) in the rodent KHT sarcoma model, either alone or in combination with single- or fractionated-dose radiation therapy.Methods: C3H/HeJ mice bearing i.m. KHT tumors were injected i.p. with ZD6126 doses ranging from 10 to 150 mg/kg. Tumors were irradiated locally in unanesthetized mice using a linear accelerator. Tumor response to ZD6126 administered alone or in combination with radiation was assessed by clonogenic cell survival assay or tumor growth delay.Results: Treatment with ZD6126 led to a rapid tumor vascular shutdown as determined by Hoechst 33342 diffusion. Histologic evaluation showed morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. When combined with radiation, a 150 mg/kg dose of ZD6126 reduced tumor cell survival 10–500-fold compared with radiation alone. These enhancements in tumor cell killing could be achieved for ZD6126 given both before and after radiation exposure. Further, the shape of the cell survival curve observed after the combination therapy suggested that including ZD6126 in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor. Finally, when given on a once-weekly basis in conjunction with fractionated radiotherapy, ZD6126 treatment was found to significantly increase the tumor response to daily 2.5 Gy fractions.Conclusion: The present results demonstrated that in the KHT sarcoma, ZD6126 caused rapid tumor vascular shutdown, induction of central tumor necrosis, tumor cell death secondary to ischemia, and enhancement of the antitumor effects of radiation therapy. [Copyright &y& Elsevier]