Synthesis and Characterization of a Theranostic Vascular Disrupting

Mar 16, 2011 - Metabolic and Molecular Imaging Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London W12 0NN, U.K...
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Synthesis and Characterization of a Theranostic Vascular Disrupting Agent for In Vivo MR Imaging Tammy L. Kalber,*,† Nazila Kamaly,†,‡ Stephanie A. Higham,† John A. Pugh,§ Josephine Bunch,§,|| Cameron W. McLeod,§ Andrew D. Miller,‡ and Jimmy D. Bell† †

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Metabolic and Molecular Imaging Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London W12 0NN, U.K. ‡ Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K. § Centre for Analytical Sciences, Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, U.K. School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. ABSTRACT: Colchicine, a known tubulin binding agent and vascular disrupting agent, causes rapid vascular shut down and central necrosis in tumors. The binding of tubulin results in tubulin destabilization, with characteristic cell shape changes and inhibition of cell division, and results in cell death. A gadolinium(III) labeled derivative of colchicine (Gd 3 DOTA 3 Colchicinic acid) was synthesized and characterized as a theranostic agent (enabling simultaneous diagnostic/real time MRI contrast imaging). In vitro, Gd 3 DOTA 3 Colchicinic acid was shown to initiate cell changes characteristic of tubulin-destabilization in both OVCAR-3 and IGROV-1 ovarian carcinoma cell lines in vitro over a period of 24 h, while maintaining the qualities of the MR imaging tracer. In vivo, Gd 3 DOTA 3 Colchicinic acid (200 mg/kg) was shown to induce the formation of central necrosis, which was confirmed ex vivo by histology, in OVCAR-3 subcutaneous tumor xenografts, while simultaneously acting as an imaging agent to promote a significant reduction in the MR relaxation time T1 (p < 0.05) of tumors 24 h postadministration. Morphological changes within the tumor which corresponded with areas derived from the formation of central necrosis were also present on MR images that were not observed for the same colchicine derivate that was not complexed with gadolinium that also presented with central necrosis ex vivo. However, Gd 3 DOTA 3 Colchicinic acid accumulation in the liver, as shown by changes in liver T1 (p < 0.05), takes place within 2 h. The implication is that Gd 3 DOTA 3 Colchicinic acid distributes to tissues, including tumors, within 2 h, but enters tumor cells to lower T1 times and promotes cell death over a period of up to 24 h. As the biodistribution/pharmacokinetic and pharmacodynamics data provided here is similar to that of conventional colchicines derivatives, such combined data are a potentially powerful way to rapidly characterize the complete behavior of drug candidates in vivo.

’ INTRODUCTION It is well established that tumor growth is dependent upon the generation of a functional vascular supply and that tumor vasculature differs significantly from that of vessels in normal tissues.14 Normal tissues have well-ordered vessels with quiescent endothelial cells, whereas tumor vasculature is a chaotic network of tortuous, thinwalled, leaky vessels, with a high proportion of immature, proliferating endothelial cells that rely on their tubulin cytoskeleton to maintain shape.3,4 Consequently, tumor endothelium can be targeted in therapeutic approaches to cancer that leave normal blood vessels unaffected.5 In particular, vascular disrupting agents (VDAs) invoke the selective collapse of tumor vasculature, leading to a loss in tumor blood flow, secondary cell death, and tumor necrosis due to ischemia.6 VDAs have largely consisted of tubulin-depolymerizing agents such as colchicine and its derivatives, and more recently, the flavonids such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). r 2011 American Chemical Society

Colchicine binds to β-tubulin, inhibiting tubulin polymerization, resulting in microtubule destabilization. Destabilization of the microtubule cytoskeleton induces cell shape changes, which in the endothelial cells of tumors, results in cell detachment, exposure of the basal lamina, and vessel occlusion.7 However, colchicine is too toxic for clinical use as it is only effective close to its maximum tolerated dose (MTD). Derivatives of colchicine have therefore been developed, the best known being disodium combretastatin A-4 3-O-phosphate (CA-4-P) and ZD6126, both of which induce vascular occlusion and therefore antitumor effects in a wide range of tumor models at doses that are less than one-tenth of the MTD.811 Received: July 21, 2010 Revised: February 17, 2011 Published: March 16, 2011 879

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Scheme 1. Synthesis of Gd 3 DOTA 3 Colchicinic acida

a

(i) 30% H2SO4, 100 °C, 5 h, 20%; (ii) DOTA-NHS-ester, DCM, 40 °C, 3 eq. Et3N, 12 h, 98%; (iii) 6H2O 3 GdCl3, H2O, 90 °C, quantitative.

The impact of VDAs on tumor tissue has been demonstrated histologically in numerous tumor models, revealing central necrosis within 24 h of a single injection with a characteristic thin rim of viable tumor cells at the periphery.9,12 The rim is thought to be a consequence of these cells receiving nutrients/oxygen from nontumor vessels in adjacent normal tissue allowing these tumor regions to regrow. Therefore, VDA treatment is not expected to cause tumor shrinkage or significant growth delay as a single therapy. Accordingly, in the absence of tumor shrinkage, there is a need for noninvasive methods that can provide an assessment of tumor vasculature and pathophysiology in response to VDA therapy in vivo. The hemodynamic tumor response to VDA therapy can be monitored by dynamic contrast enhanced magnetic resonance imaging (MRI) using gadolinium(III) (Gd) chelates,1317 as well as by intrinsicsusceptibility MRI, which is sensitive to changes in blood oxygenation.18,19 Otherwise, pathological changes have been observed by MR methodologies such as diffusion-weighted MRI2022 or necrosis-avid contrast agents capable of detecting tumor necrosis,23 as well as methods to investigate apoptosis and cell death, such as choline spectroscopy24 or targeted contrast agents.25 Since all these imaging methodologies are responding to secondary changes at selected time points after VDA administration, then this suggests that VDA imaging itself could provide the means not only to reach a better understanding of drug biodistribution/pharmacokinetics, routes of clearance, etc., but also to simultaneously monitor pharmacodynamic phenotypic effects in real time as well as tumor necrosis in particular. Previously, colchicine has been conjugated with imaging agents2631 including Gd-chelates,32 but mainly for applications in vitro or in terminal in vivo studies. The aim of the study reported here was to conjugate an MRI contrast agent to colchicine in order to produce a novel theranostic VDA capable of remaining as a functional VDA as well as acting as a competent contrast agent for similtaneous diagnostic/real time MRI contrast imaging in vivo. Here, we describe the synthesis and characterization of such a theranostic VDA followed by a range of in vitro and in vivo imaging studies over 24 h post-VDA administration, concluding with histology and laser ablation inductively coupled mass spectrometry (LA-ICP-MS) to correlate Gd (III) distribution with associated relaxation time (T1) changes shown by in vivo MR.

’ EXPERIMENTAL SECTION Gerneral procedures. All chemicals were of analytical/high grade and purchased from Sigma-Aldrich (Dorset, U.K.) or Macrocyclics (Dallas, USA). Mass spectrometry (MS) were performed using VG-070B, Joel SX-102, or Bruker Esquire 3000 electrospray mass spectrometry (ESI) instruments. 1H NMR spectra were recorded on a 400 MHz Bruker Advance 400 spectrometer. All MRI experiments were conducted on a 4.7 T Varian Direct Drive MRI scanner (Varian Inc., Palo Alto, CA, USA). All procedures on animals were carried out in accordance with U.K. Home Office regulations and the Guidance for the Operation of Animals (Scientific Procedures) Act (1986). Chemical Synthesis. Colchicinic acid (Scheme 1) was synthesized according to the previously reported method by Satpati et al.26 Tetraazacyclododecanetetraacetic acid (DOTA) in the form of DOTA 3 NHS ester (100 mg, 0.121 mmol) and colchicinic acid (41.55 mg, 0.121 mmol) were added to an air-evacuated roundbottom flask, and to this, dry dichloromethane (30 mL) followed by triethylamine (3 eq 0.363 mmol) was added and stirred at 45 °C for 12 h. The solvent was removed and the crude mixture purified by flash column chromatography using Merck 0.040 to 0.063 mm, 230 to 400 mesh silica gel, and the product eluted with dichloromethane/methanol/acetic acid (6:3:1 v/v). The collected fractions were reduced to yield a solid (87 mg, 98%) DOTA 3 Colchicinic acid (Scheme 1). Rf [dichloromethane/methanol/acetic acid, 6:3:1 v/v] 0.11. 1H NMR (400 MHz, MeOD) 8.28 (s, br, 1H, OH, H10), 7.76 (s, 1H, H8), 7.46 (s, 1H, H11), 7.11 (d, 1H, H12), 6.92 (d, 1H, H4), 6.48 (s, br, 1H, CNHCOCH2), 4.34 (d, 2H, NCOCH2N), 3.64 (m, 16H, 4 NCH2CH2N), 3.26 (m, 6H, 3 NCH2COOH), 3.12, (s, 3H, -OCH3, H1), 3.08 (m, 4H, CH2CH2, H5,6), 2.77 (s, 3H, OCH3, H2), 2.63 (s, 3H, OCH3, H3). MS (ESIþ) calculated for C35H47N5O12 m/z 729.7740; found, 730.10 (M þ H)þ. A stoichiometric amount of gadolinium chloride (20.90 mg, 0.056 mmol) was added to DOTA 3 Colchicinic acid (41.06 mg, 0.056 mmol) and stirred in doubly distilled water (20 mL) at 90 °C overnight. The water was freeze-dried to yield a powder (50 mg, quantitative yield): Gd 3 DOTA 3 Colchicinic acid (Figure 1). Rf [dichloromethane/methanol/acetic acid, 6:3:1 880

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When tumors reached approximately 710 mm3, mice were anesthetized with an isoflurane/O2 mix and scanned as described for phantoms using the parameters; 5 TRs ranging from 4005000 ms; TE = 15 ms; FOV = 45  45 mm; averages, 1; matrix size, 256  128, 2.0 mm thickness; and 20 consecutive transverse slices covering the whole abdomen. Mice were removed from the magnet and a tail vein cannulated for the administration of 200 mg/kg (22.64 mmol) Gd 3 DOTA 3 Colchicinic acid (n = 4), 22.64 mmol DOTA 3 Colchicinic acid (n = 4), 22.64 mmol Gd 3 DOTA (n = 4), or 200 μL of saline (n = 4); 200 mg/kg was used as this is an optimal dose that has been used for both CA-4-P and ZD6126 in mice. Mice were then rescanned with the same parameters 2, 8, and 24 h post-injection. After the final scan mice were sacrificed, the tumor was excised and frozen for LA-ICP-MS and hematoxylin and eosin (H & E) staining. The liver and kidneys were also taken for H & E only. MRI Data Analysis. A region of interest (ROI) was drawn around the whole tumor excluding the skin on MR images using Image J (National Institute of Health, USA).34 Mean signal intensities of the ROIs at different TR values were measured and used to calculate the MR transverse relaxation time T1 using Graphpad Prism (San Diego, California, USA). Mean T1 values for each cohort were derived and a two-tailed unpaired t test assuming equal variances performed at each time point to determine significant difference, with a 5% level of statistical significance. Circular ROIs were then drawn in the liver and kidney to assess clearance and the signal intensities converted to T1 as described above. LA-ICP-MS. Frozen tissue was mounted onto a cryostat chuck using water/ice slush frozen to 20 °C and sections cut at 20 μm using a cryotome. The laser ablation system (LSX-200, Cetac, 266 nm Nd:YAG) was configured to perform multiple line rasters, generating 2D elemental distribution maps. A beam diameter of 50 μm was used for ablating tumor sections (with energy 0.6 mJ), a scanning speed of 50 μm/s and laser frequency of 10 Hz. For ICPMS (Agilent, HP4500 Series 100), the elements Gd157 and Zn66 (an element widespread in biochemistry) were monitored in a timeresolved mode, isotopes were selected on the basis of highpercentage abundance, and minimal isobaric and polyatomic interferences. Tumor sample areas were in the range 130190 mm2. Elemental maps were produced using the Graphis software package (Kylebank Software Ltd., Ayr, UK). Adjacent sections where taken for H & E staining. Histological Assessment. Sections were incubated with hematoxylin solution for 1 min, and then in freshly prepared eosin Y (both Sigma) for 30 s. The sections were then dehydrated with alcohol and passed through histoclear (Fisher Scientific), mounted with DPX (VWR), and imaged using the Olympus IX71 microscope as described in the toxicity studies.

Figure 1. In vitro cell studies of DOTA 3 Colchicinic acid in OVCAR-3 cells. A histology picture of OVCAR-3 cells, (A) without and (B) with 1 mM 24 h after DOTA 3 Colchicinic acid; (C) cell counts for both colchicine (white) and DOTA 3 Colchicinic acid (gray) after 24 h at various concentrations. Magnification 100.

v/v] 0.18. MS (ESIþ) calculated for C35H44GdN5O12 m/z 884.00; found, 885.23 (M þ H)þ. The gadolinium compound was characterized by ESI-MS, and the presence of free gadolinium ions was determined by the xylenol orange test previously described by Barge et al.33 In Vitro. Phantom Studies. 0.5 mM of Gd 3 DOTA 3 Colchicinic acid, DOTA 3 Colchicinic acid, Gd 3 DOTA (Dotarem, Guerbet, France), or water was used to assess signal enhancement. A range of 6 concentrations (0.030.5 mM) of Gd 3 DOTA 3 Colchicinic acid or Gd 3 DOTA was then used to calculate relaxivity for each contrast agent. Phantoms were placed in a quadrature 1H volume coil and positioned into the MRI scanner. A spinecho sequence with the following parameters was used to assess T1: 10 TRs ranging from 50 to 7000 ms; TE = 10 ms; FOV = 45  45 mm; averages, 1; matrix size, 256  128 and a 2.0 mm thickness; and bore temperature, ∼ 20 °C. Tissue Culture. Human ovarian carcinoma OVCAR-3 and IGROV-1 cell lines were grown in T175 flasks (Fisher Scientific, Loughborough, UK) in Dulbecco’s modified Eagle's medium (DMEM) (Sigma), supplemented with 10% heat inactivated fetal calf serum (GIBCO, Grand Island NY, USA) in an incubator at 37 °C with 95% air and 5% CO2. Cells were grown to 100% confluence before being trypsinized, counted, and then either plated for in vitro experiments or prepared for subcutaneous implantation. Toxicity Studies. Cells were plated at 4  105 in 6 well plates 24 h prior to incubation with a range of concentrations (1 nM1 mM) of either DOTA 3 Colchicinic acid or colchicine dissolved in serum free DMEM. Histological morphology of cells was recorded at 2 h intervals up to 8 h, and at 24 h using an Olympus IX71 microscope (Olympus, Middlesex, UK). The cells were harvested at the 24 h time point and the numbers counted using a hemocytometer. Gd 3 DOTA 3 Colchicinic Acid Uptake. Cells were plated at 1.5  105 in T25 flasks 24 h prior to the incubation of 1 mL of 1 mM Gd 3 DOTA 3 Colchicinic acid (the most efficient incubation dose from toxicity studies). Cells were then washed, harvested, and pelleted and resuspended in 1% agarose (Sigma, UK) at 2 h intervals up to 8 h, and at 24 h after incubation and scanned as described for phantoms. In Vivo. MRI. OVCAR-3 cells (5  106/0.1 mL) were inoculated into the flank of 68 weeks old Balb/c nude mice. Once palpable, tumors were measured in three orthogonal dimensions every 2 days up until treatment, then pre and 24 h post-treatment, using calipers. Tumor volumes were estimated assuming an ellipsoid shape using the formula volume = length  width  depth  π/6.

’ RESULTS Chemical Synthesis. The synthesis of Gd 3 DOTA 3 Colchicinic acid was carried out in three steps as shown in Scheme 1. The control compound DOTA 3 Colchicinic acid was obtained in good yield and was characterized by 1H and MS spectroscopy. Complexation of Gd(III) within DOTA could not be confirmed by NMR due to the paramagnetic nature of Gd(III) and was therefore confirmed by ESI-MS and the xylenol orange assay. Isotopic peaks of Gd(III) were highly visible in the MS trace, and there were no significant free Gd(III) ions detectable after complexation according to the xylenol orange assay, thereby confirming the formation of Gd 3 DOTA 3 Colchicinic acid. 881

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Figure 2. A set of two contiguous T1 MRI images acquired from similar anatomic points from the same mouse bearing a subcutaneous OVCAR3 tumor pre, 2, 8, and 24 h after 200 mg/kg DOTA 3 Colchicinic acid or Gd 3 DOTA 3 Colchicinic acid administration. Column 24 h (annotated) shows the enlarged 24 h tumor image, which includes annotated areas of signal change in the Gd 3 DOTA 3 Colchicinic acid treated tumors. The white arrow denotes a phantom placed to correlate images.

In Vitro. Phantoms showed that Gd 3 DOTA 3 Colchicinic acid achieved a 96% reduction in T1 compared to the same concentration of DOTA 3 Colchicinic acid or water, and its relaxivity was calculated at 3.83 mM1s1. This was equivalent to that of nonconjugated Gd 3 DOTA, which was calculated at 3.66 mM1s1. The efficiency of DOTA 3 Colchicinic acid for binding tubulin was assessed in two ovarian carcinoma cell lines. Normally, endothelial cell lines such as HUVEC’s are used to assess the functionality of novel VDAs. However, the effects of tubulinbinding drugs are not specific to endothelial cells. Indeed, some tumor cell types are more sensitive to VDA tubulin depolymerization than endothelial cells.35 The extent to which direct tumor cell death has contributed to VDA therapy is likely dependent on tumor type. Therefore, DOTA 3 Colchicinic acid was tested in tumor cells to assess tubulin binding and tubulin destabilization activities in order to obtain a better idea of the amount of cell death/antitumor effect that Gd 3 DOTA 3 Colchicinic acid may provide in vivo, in addition to VDA action. Cell counts showed that colchicine was effective at significantly reducing cell numbers at all concentrations in both the OVCAR-3 and IGROV-1 cell lines, whereas DOTA 3 Colchicinic acid was only effective in the millimolar range, causing reductions in cell numbers in both cell lines (Fgure 1C). These high concentration effects were the same for the Gd(III) derivate (Gd 3 DOTA 3 Colchicinic acid) and correlated well with colchicine effects at the same high concentrations. Arguably, these data suggest that Gd 3 DOTA 3 Colchicinic acid may be much less cytotoxic than colchicine to cells in general but retains colchicine's drug-action mechanism in spite of the conjugated Gd 3 DOTA chelate (Figure 1C). Light microscopy demonstrated the emergence of initial cell shape changes as early as 2 h post-treatment with DOTA 3 Colchicinic acid, followed by rounding of the cells, and then detachment from the flask walls at 24 h (Figure 1A and B). MR images of cell pellets taken at specific time points after incubation with Gd 3 DOTA 3 Colchicinic acid (1 mM) showed a 54% reduction in T1 at 2 h compared to that in control cells, which decreased further reaching a maximum of

Figure 3. T1 (ms) measured pre, 2, 8, and 24 h post-injection of 200 mg/kg (22.64 mmol) Gd 3 DOTA 3 Colchicinic acid, 22.64 mmol DOTA 3 Colchicinic acid, or 200 μL saline for (A) tumor, (B) liver, and (C) kidney. Data points ( standard error of the mean (n = 4 in each group). * = P < 0.05.

83% reduction at 6 h. After 6 h, T1 recovered slightly, but at no point returned to baseline over the 24 h period (data not shown). These data are in accordance with the histology data and suggest that initial tubulin binding takes place in vitro within the first 2 h of incubation, followed by further cellular uptake and tubulin binding over 6 h. In Vivo. Mice showed no adverse effects throughout the study. There was no significant difference in tumor size between cohorts, consistent with the fact that colchicine and its derivatives do not typically cause tumor shrinkage or growth delay. Images of pretreated tumors appeared to be homogeneous in presentation. At 24 h, the tumor images showed evidence of morphological changes mainly evident in the central area of the tumor as areas of low signal intensity (dark areas) in 3 out of 4 of the Gd 3 DOTA 3 Colchicinic 882

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Figure 4. Laser ablation inductively coupled mass spectrometry data of Gd157 and Zn66 distribution in tumor sections of OVCAR3 tumors 24 h after 200 mg/kg Gd 3 DOTA 3 Colchicinic acid or 200 μL of saline administration.

acid treated mice (Figure 2). These changes in signal intensity were not observed at any time point, in any other cohort throughout the study. Changes in mean T1 are presented (Figure 3) for the tumor, liver, and kidney. Gd 3 DOTA exhibited similar values and trends as saline and DOTA 3 Colchicinic acid controls; therefore, these data have been omitted (from Figure 3). Tumor T1 presented no change in mean T1 from the baseline for any cohort at 2 h. A slight but nonsignificant reduction of 3% in mean T1 was observed at 8 h postGd 3 DOTA 3 Colchicinic acid administration, which decreased further to become a significantly (p < 0.05) greater reduction of 6% in mean T1 at 24 h post-administration in comparison with that in saline, Gd 3 DOTA, and DOTA 3 Colchicinic acid control cohorts (Figure 3A). A significant (p < 0.05) reduction of 55% in mean T1 was also observed in the liver post-Gd 3 DOTA 3 Colchicinic acid administration at 2 h in comparison to that in saline, Gd 3 DOTA, or DOTA 3 Colchicinic acid control cohorts. This reduction in mean T1 was sustained throughout, up until 24 h post-administration (Figure 3B). In the case of the kidney, no single significant change in T1 was observed in any cohort (Figure 3C). LA-ICP-MS. Elemental maps of Gd157 and Zn66 distribution in tumors against a saline control were generated 24 h post-Gd 3 DOTA 3 Colchicinic acid administration (Figure 4). LA-ICP-MS analysis indicated substantial amounts of Gd(III) present throughout the whole tumor. Using Zn66 as an indication of viable tissue, it can be clearly seen that the largest accumulation of Gd(III) takes place in complementary areas that may be necrotic. This suggests that Gd(III) has accumulated in tumor zones that were originally well perfused with blood vessels but became necrotic due to the occlusion of blood vessels post-VDA action. Our data indicate that the MRI-active VDA is still trapped within these apparently necrotic areas. Histology. H & E staining showed that all tumors 24 h post-Gd 3 DOTA 3 Colchicinic acid or DOTA 3 Colchicinic acid administration

possessed a large central core of necrosis with a rim of viable tumor tissue at the tumor periphery. Therefore, the degree of necrosis was not dependent on Gd(III) incorporation (Figure 5B). In contrast, tumors 24 h post-saline and Gd 3 DOTA administration possessed minimal necrosis and exhibited large areas of viable tumor tissue throughout the tumor (Figure 5A). Otherwise, the liver, which was also observed to accumulate substantial amounts of Gd(III) postGd 3 DOTA 3 Colchicinic acid as evidenced by substantial reductions in mean T1 values (Figure 3B), showed no significant differences in liver morphology between animals treated with saline or any agent used in this work (Figure 5C and D). The kidney too was substantially unaffected by Gd 3 DOTA 3 Colchicinic acid administration or the administration of any other control compounds (data not shown).

’ DISCUSSION In this study, we have synthesized and characterized a novel theranostic tubulin binding agent capable of working as a vascular disrupting agent that was capable of both reducing T1 as well as keeping its efficacy to bind to tubulin in vitro, resulting in cell shape changes and central tumor necrosis in vivo characteristic of VDA treatment that could be followed with standard MR sequences longitudinally. The conjugation of DOTA to colchicine resulted in DOTA 3 Colchicinic acid, which was complexed with gadolinium to produce the novel compound Gd 3 DOTA 3 Colchicinic acid. In vitro results showed that conjugation of Gd 3 DOTA to colchicine did not affect the relaxivity of the contrast agent as an MR probe. DOTA 3 Colchicinic acid was also found to be a functional therapeutic agent and was effective at causing characteristic VDA changes in both ovarian cell lines in vitro at concentrations of the millimolar range, suggesting that cells were less sensitive to the effects of cytotoxicity as afforded by unconjugated colchicine. 883

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24 h after treatment with Gd 3 DOTA.Colchcinic acid. As there was no change in T1 for the saline or DOTA 3 Colchicinic acid cohorts, the decrease in T1 relaxation is purely due to Gd 3 DOTA 3 Colchicinic acid acting as a contrast agent rather than effects caused by therapeutic effect, as these were not apparent in the nongadolinium derivative, which presented necrosis scores similar to that of Gd 3 DOTA.colchicinic acid by histology. Although Gd 3 DOTA also contains gadolinium, the nature of the molecule means that it has a short half-life and rapid clearance; therefore, the fact that there was no change in T1 for this cohort suggests washout within the initial 2 h period. Morphological changes corresponding to necrosis were detected within the tumor at 24 h on MR images in the Gd 3 DOTA.Colchcicinic acid cohort only. Darkening of areas within the originally homogeneous tumor presentation were detected mainly within the center of the tumor and correlated well with the degree of necrosis by histology. LA-ICP-MS data revealed high concentrations of gadolinium throughout the tumor. Using Zn66 as a marker of viable tissue shows that areas assumed to be necrotic have high degrees of gadolinium present and therefore may represent previously well vascularised areas where Gd 3 DOTA 3 Colchicinic acid is still bound within the occluded vessels. The increased contrast between what is believed as the necrotic core and viable rim on images for the Gd 3 DOTA 3 Colchicinic acid cohort is most likely due to perfusion in the presence of bound Gd 3 DOTA 3 Colchicinic acid and is enough to enhance the signal intensity on T1 images, whereas in areas of necrosis and increased paramagnetic substances due to therapy, such as increased permeability, platelet aggregation, coagulation, and hemorrhagic necrosis, it causes darkening on the image and therefore increases contrast between the two areas.40 The liver also showed signal attenuation due to Gd 3 DOTA 3 Colchicinic acid accumulation, whereas no changes were observed in kidney T1. As histology did not show any structural abnormalities and therefore no cyctoxicity to normal tissue in either organ, this may indicate the route of excretion as being via the liver and bile, which has also been shown for ZD6126 when tagged with carbon 14.41,42 The therapeutic effect of Gd 3 DOTA 3 Colchicinic acid is therefore comparable to changes induced by both CA-4-P and ZD6126 by MR where CA-4-P studies have shown early changes in signal with recovery to baseline at 24 h in both animal models and humans at clinically relevant doses,17 whereas ZD6126 studies show no evidence of tumor recovery at 24 h16,43 or up to 96 h after administration,43 suggesting as with the in vitro data that the Gd 3 DOTA.Colchicine compound binds irreversibly in a similar manner to ZD6126.38 As well as carbon 14, colchicine has previously been conjugated to a wide range of imaging tracers, including Gd 3 DOTA, using the same demethylation of the methoxy group adjacent to the carbonyl.2632 These studies are mainly in vitro or if in vivo have required animal sacrifice at each end point, such as the radiolabeled studies,26,30,31,41,42 and therefore do not provide longitudinal in vivo data. Studies that retained the toxicity of colchicine such as studies by Clark et al. evaluated the binding and subsequent cell changes in vitro but could not be used in vivo.27 In other studies that reduced the toxicity of colchicines, although showing increased uptake and retention in vitro32 and in vivo,26 were unclear as to whether there was any significant therapeutic response.26 In conclusion, we have successfully synthesized and characterized a novel derivative of colchicine that is capable of functioning as a competent imaging tracer and as a therapeutic tubulinbinding agent in vitro and in vivo. The utilization of this has allowed for in vivo imaging of central necrosis as a therapeutic

Figure 5. Hematoxylin and eosin stained sections of OVCAR-3 tumors (A,B) and normal liver parenchyma (C,D) 24 h after the administration of (A and C) saline and (B) 200 mg/kg Gd 3 DOTA 3 Colchicinic acid. Magnification is 100.

Colchicine is too toxic for clinical use and is effective only at or close to its MTD, where direct cell cytotoxicity is the principal mechanism of action. A reduction in tumor cell cytotoxicity, therefore, suggests that DOTA 3 Colchicinic acid has a lower MTD and therefore a wider therapeutic window than colchicine, which would be well tolerated in animals. DOTA 3 Colchicinic acid was tested in ovarian carcinoma cells initially in vitro instead of endothelial cells, as rapidly dividing tumor cells are also susceptible to VDA therapy and would give a better understanding about the secondary effects of VDA therapy on tumor cells that would be on top of the initial effect of vascular shut down.35 Initial cell shape changes were observed at as early as 2 h after treatment with DOTA 3 Colchicinc acid, followed by cell rounding caused by the contraction of the tubulin cytoskeleton, decreasing intracellular adhesion, and subsequent cell loss due to detachment, similar to what has been shown in endothelial cells after doses of either ZD6126 or combretastatin derivatives in vitro.9,36,37 Gd 3 DOTA 3 Colchicinic acid also caused T1 reductions in cells in vitro at 2 h, which further decreased over 6 h. After 6 h, the T1 recovery may be due to lower numbers of cells or changes in cell shape effecting water diffusion. However, as the T1 does not recover completely, this implies that binding is irreversible as with colchicine itself,38 resulting in long drug exposures that will therefore cause cell cytotoxicity, which would lead to increased tumor cell death.39 As expected for single dose VDA treatment, Gd 3 DOTA 3 Colchicinic acid did not cause tumor shrinkage or growth delay. Reductions in T1 were apparent at 8 h and became significant at 884

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response to VDA therapy in tumors in real time and has been validated by both histology and LA-ICP-MS. This has given a better understanding of the timing of necrosis and has strengthened the requirement for smart probes to be incorporated into the experimental design for in vivo applications.

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’ AUTHOR INFORMATION Corresponding Author

*Tel: þ44 208 383 3497. Fax: þ44 208 383 1511. E-mail: tammy. [email protected].

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