Abstract: Two new porphyrin-based MR imaging contrast agents, Gd-hematoporphyrin (Gd-H) and Gd-tetra-carboranylmethoxyphenyl-porphyrin (Gd-TCP) were synthesized and tested in mice with human colorectal cancer cells (HT29/219) as new colorectal contrast agents. Human colorectal cancer cells (HT29/219) were studied in 40 (eight groups of five) mice. The effect of different contrast agents (Gd-TCP, Gd-H, GdCl3 and Gd-DTPA) on proton relaxation times was measured in tumor and other organs. The biodistribution, T1 values, signal enhancement and the Gd concentration for different contrast agent solutions are investigated and the results are compared. A reduction of 15 and 12% in T1 values was revealed 24 h after injection of Gd-H and Gd-TCP, respectively. The percent of Gd that localized to the tumor measured by UV spect was approximately 55% for Gd-H and 45% for Gd-TCP which was higher compared with control (GdCl3) after 24 h. Signal enhancement of 5.6 and 4.5% over the control was observed for Gd-H and Gd-TCP, respectively. The high concentration of Gd in the tumor is indicative of a selective retention of the compounds and indicates that Gd-H and Gd-TCP are promising MR imaging contrast agents for colorectal cancer cell detection. Gd-porphyrins have considerable promise for further diagnostic applications in magnetic resonance imaging.
INTRODUCTION
The development of contrast agents with tissue-specific enhancement deserves considerable attention because of their potential in earlier and improved diagnosis and in possible therapy. One approach to increasing the specificity of MR image contrast is to use porphyrin-based agents (Ogan et al., 1987; Furmanski and Longley, 1988; Ni et al., 1997).
Porphyrin-based agents are in the investigation stage (Oagan et al., 1987; Young et al., 1994; Miura et al., 1996; Bourre et al., 2003). A related class of organic molecules called texaphyrins, which is a modified porphyrin, has recently received considerable interest for its high tumor selective uptake (Young et al., 1994). The gadolinium complex of texaphyrin is a-selective radiation sensitizer that is detectable at MR imaging (Young et al., 1994). The porphyrins chosen for this study are natural porphyrins, hematoporphyrin [8, 13-bis (hydroxyethyl) -3, 7, 12, 17-tetramethyl 21H, 23H-porphine-2, 18-dipro- pionic acid] and a synthetic boronated porphyrin, 1, 6, 11, 16-tetra (3-o-carboranyl-methoxy) phenyl-porphyrin (TCP). The beauty of porphyrins is that these materials offer not only a stable chelate for the transportation of paramagnetic metals into the tumors, but also the potential attachment of molecules that could destroy the cancer cell. The synthetic porphyrin TCP is an example of this concept where boron atoms have been attached chemically to the porphyrin, thus offering the potential for boron neutron capture therapy (BNCT).
In vivo studies of Gd-DTPA-monoclonal antibody and Gd-porphyrins as MR imaging contrast agents for tumor detection have been investigated in mice (Shahbazi-Gahrouei et al., 2001a, 2002). The two porphyrin-based agents, TCP and hematoporphyrin, can be used simultaneously as MR imaging contrast agents by choosing gadolinium as the metal for incorporation. The synthesis of these compounds has been described previously (Shahbazi-Gahrouei et al., 2001a, 2001b). In this study, Gd-H and Gd-TCP are injected into mice with a colorectal cell line (HT29/219). The pharmacokinetic, signal intensity, T1 relaxation times and Gd concentrations of the contrast agents are presented and the results are compared for the first time.
MATERIALS AND METHODS
Chemical: The porphyrin-based MR imaging contrast agents were prepared as described previously (Shahbazi-Gahrouei et al., 2001a, 2001b). Briefly, hematoporphyrin ([8, 13-bis (1-hydroxyethyl)-3, 7, 12, 17-tetramethyl-21H,23H-porphine-2, 18-dipropionic acid]) powder was suspended in distilled water and was added to the gadolinium solution and refluxed until the solution become homogeneous to yield Gd-H. The synthesis of Gd-TCP involved the formation of an aldehyde containing the o-carborane group. This aldehyde was reacted with pyrrole to form the porphyrin, TCP-H2. The gadolinium ion was inserted into TCP-H2 by adaptation of Muira’s (1996) method for the nickel complex.
Solutions of Gd-H and two discrete agents (GdCl3, Gd-DTPA) were prepared by accurately dissolving the required amount in 0.9% saline solution. Gd-TCP (15 mg, 0.010 mmol) was dissolved in 1 mL of cremophor EL (CRM) and 2 mL of 1,2-propanediol. This solution was transferred into a 10 mL volumetric flask and a 0.9% saline solution was added to the mark. This gave a final concentration of 1.0 mM.
Animals: The animal studies were performed with 40 mice of 6-8 week old with a mean weight of 27 g (Animal house, Faculty of Medicine). Animals were randomly divided into five groups of eight. Each group was housed in cages in a humidity and temperature controlled.
Cells: The human colorectal cells, HT29/219, was purchased (Pasteur institute, Tehran), in flask and injected (2.5x106 cells) subcutaneously into both flanks of studied mice.
Injected dose: Two to three weeks after cancer cell inoculation, when the tumor diameter was 2-4 mm, mice were injected with the different contrast agent conjugates. All contrast agents were diluted in physiological saline to a final concentration as injected in bolus doses (0.01 mmol kg-1 of body weight). Five groups of five mice received an intraperitoneal (i.p.) injection of Gd-H. Two groups received Gd-DTPA or Gd-TCP and the last group received GdCl3, being the control group. The injected volume was 250 μL. The animals were sacrificed by an over-dose of pentobarbital sodium at 24 h post i.p. injection, followed by removal of tumor, kidney and liver. To investigate of biodistributions of Gd-H four groups of five mice were also sacrificed at 7, 12, 48 and 72 h post i.p. injection.
Sample preparations: All samples were prepared using an acid digestion procedure according to the method of Tamat et al. (1989). Briefly, to a weighed sample of tissue (60-120 mg) in a polyethylene vial, 0.3 mL of 72% perchloric acid was carefully added and the contents swirled to mix. 0.6 mL of 32% hydrogen peroxide was added and the vials placed in the shaking bath for 5 h at 23°C. At this stage, the vial contents were clear and colourless. The samples were diluted to 3 mL of distilled water and filtered through a 0.45 μm Millipore filter before being introduced into both NMR and UV-spect. experiments.
Sample analysis: The T1 relaxation times and signal intensity of solutions was measured using an Inversion Recovery (IR) pulse sequence technique by 11.4 T NMR (Bruker instrument, Tarbiat Modarres University, Tehran). The values of echo time and repetition time were optimized for different solutions. The UV-spectrophotometer (Dunmow Essex, Jenway Ltd., UK) was used to measure the Gd content of tissue.
The T1 relaxation time and signal intensity is measured using an Inversion Recovery (IR) sequence (180° rf-τi-90° rf-collect FID). The repetition time chosen was 2.5 times the estimated T1. Ten inversion delays (TI) were used with each increasing by a factor of two. The minimum inversion time used was approximately one-tenth of the estimated T1 value.
RESULTS
General aspects: All mice tolerated the procedures well, including tumor growth and response to contrast agents. No adverse effects were observed after injection of contrast agents and no animal death was recorded during tumor growth or post injection.
Pharmacokinetics of Gd-H: At what time and to what extent the MR imaging can be obtained depends on the accessibility of contrast agents to the tumors. For this reason, Gd content of selected tissues was measured after sacrificing of animals at different time (7, 12, 24, 48 and 72 h) the results being shown in Fig. 1. As this results illustrated, the maximum Gd content for tumor uptake occurs at 24 h post injection of Gd-H and this time was selected as sacrificed time for further animal study.
T1 relaxation times: The effect of contrast agents on proton relaxation times (T1) was measured in tumors and the other harvested organs. Table 1 shows the T1 measurements of organs using different contrast agents and untreated mice (control). The plot of T1 values for removed tissues is shown also in Fig. 2.
These result, reflect the gadolinium concentrations in the tissues reported above. The high uptake of Gd-H and Gd-TCP by the tumor resulted in approximately 15 and 12% change in T1 relaxation time of the water in human colorectal cell when compared to the T1 value for the control. Clinically agent of Gd-DTPA, showed a 3.3% decrease in the T1 value for the tumor relative to control.
| Fig. 1: | The biodistribution of Gd-H in selected tissues of mice (N = 5) |
| Fig. 2: | T1 relaxation times of gadolinium compounds in tumor and in selected organs of mice (N = 5) |
| Table 1: | Average T1 relaxation times (msec)a in tumor and other harvested organs of mice with human colorectal cell (HT29/219)* |
| *: Tissues were removed 24 h after injection of different contrast agents. a: Data are mean±SEM of values obtained from five mice | |
MR image signal intensity: The graphs of MR image signal intensity for tumor and selected organs and different contrast agents are shown in Fig. 3. The enhancement of the MR image signal intensity after injection of different contrast agents causes the changes in T1 values. The biggest signal intensity was observed for the tumor upon injection of porphyrin based agents, reflecting the shortening of T1 relaxation times and the maximum accumulation of the contrast agent in the tumor. In the control, the MR image signal intensity for tumor was slightly lower than that recorded for the Gd-porphyry agents. This may result from the T1 relaxation time for control (GdCl3) being longer than the porphyrin based agents, which decreases signal intensity and is consistent with T1 values (Table 1).
| Fig. 3: | MR image signal intensity of tissues 24 h after injection of different gadolinium compounds (N = 5) |
| Fig. 4: | Comparison of biodistribution of the gadolinium uptakes in colorectal cells in mice for tumor and selected organs after 24 h post injection (N = 5) |
Gadolinium content of tissues: Figure 4 clearly show that the biggest concentration of gadolinium in the tumor was achieved using Gd-H and Gd-TCP (55 and 45%) compared to control (GdCl3). For Gd-DTPA, the Gd uptake by the tumor was slightly smaller than that of porphyrin based agents which indicated the attachment of gadolinium ions into the tumor.
DISCUSSION
Quantitative determination of paramagnetic contrast agents in tissues will help to obtain optimal MR image intensity (Wedeking et al., 1992). Two methods have been employed to determine the relative concentration of contrast agents in tissues following administration. Most commonly, the information is obtained from relative MR image intensity changes. This method suffers from pure accuracy because the relaxivity of the agents in the tissues is generally unknown. The more direct and accurate method involves sacrifice and dissection of the animals following contrast agents administration. Concentration is then determined by UV spectrophotometer.
Paramagnetic chelates using the endogenous porphyrin ring as the chelating agent are a promising and interesting family group of potential MR imaging contrast agents (Ferrand et al., 2003). Gd-porphyrins was synthesized and investigated. By choosing gadolinium as the metal for incorporation into the TCP and hematoporphyrin, they used as MR imaging contrast agents. It is possible to achieve shortening of T1 in the tumor by means of gadolinium incorporation into two studied porphyrin based agents.
Pharmacokinetics of Gd-H: At 24 h post injection, the gadolinium content of Gd-porphyrins was greater in the tumor than that of other selected organs (Fig. 1). In this study, the optimum time to sacrifice animals occurred at 24 h post i.p. injection. Other researchers (Lyon et al., 1987; Matsumara et al., 1994) also found that the enhancement effect of Gd agents on MR imaging continued for 24 or 72 h post injection. The result showed a good accumulation of the porphyrin based agents in tumor at 24 h post injection which are in good agreement with literature (Lyon et al., 1987; Matsumara et al., 1994). This means that Gd-H and Gd-TCP are suitable as a diagnostic colorectal contrast agent for MR imaging. Improvement of MR imaging techniques and novel contrast agents can also help to MR imaging as a powerful diagnostic modality for malignant and benign tissues.
The liver retained high amount of gadolinium for the Gd-porphyrins. Some of the gadolinium found in the liver might represent gadolinium dissociated from the porphyrin. It is known that free gadolinium accumulates in the liver and this may explain some of the high uptake in the reticuloendothelial organ (Haley, 1965). This was in agreement with results of gadolinium content observations reported by other researchers (Lyon et al., 1987; Ni et al., 1997). Significant gadolinium accumulation in the liver and kidney probably reflected clearance and metabolism of the gadolinium complex (Koenig, 1991).
As these results indicate, the lowest amount of gadolinium was observed in the kidney at 24 h post injection. This is due to the properties of gadolinium-based contrast agents, which are hydrophilic and accumulated in the extracellular water of the tissues and have rapid renal excretion, which is consistent with rapid clearance (Koenig, 1991).
T1 relaxation times: As can be seen from Fig. 2, all contrast agents show shortening T1 relaxation time in the liver, except for GdCl3. The most significant decrease in T1 relaxation times of the liver occurred when treated with porphyrin compounds. These results are consistent with the gadolinium concentrations found in this organ.
The decreases in the T1 values of the other discrete gadolinium complexes (GdCl3 and Gd-DTPA) were in line with the concentrations of gadolinium absorbed by the tumors. These reductions in T1 values of the tumor upon addition of contrast agent are highly significant, even though this concentration of agents used in this study (0.005-0.01 mmol kg-1) is much lower than the doses of Gd-DTPA commonly used as a contrast agent in clinical MR imaging (0.1 mmol kg-1).
Figure 2 also illustrated that the greatest T1 relaxation time was observed in the kidney after administration of GdCl3. This is probably due to free gadolinium and its urinary excretion. No significant change in T1 value was observed for the other contrast agents.
MR imaging signal intensity: As Fig. 3 showed the biggest signal intensity was observed for the tumor upon injection of Gd-H (5.6%) and Gd-TCP (4.5%) over control, reflecting the shortening of T1 relaxation times and the maximum accumulation of the contrast agent in the tumor. The enhancement effect of the porphyrin complexes in this study is in good agreement with that reported previously by conjugation of Gd-DTPA with porphyrins under in vivo conditions in mice (Hindre et al., 1993). The results also showed that the enhancement effect of the image intensity of the tumor following administration of Gd-DTPA shows the potential application of this contrast agent.
These results also demonstrated the MR imaging contrast-enhancing capabilities of Gd-TCP. This signal enhancement of Gd-TCP suggests the delivery of boron atoms into the cancer cells. Then Gd-TCP could be a dual use of the compound, as it can enhance contrast between tumor and normal tissues in MR images and be potentially effective as an agent for Boron Neutron Capture Therapy (BNCT).
Gadolinium content of tissues: The gadolinium concentration in tumor and other tissues was measured by UV spect using an acid digestion procedure and results showed in Fig. 4. A higher concentration of Gd was achieved in tumor as compared with discrete compounds (GdCl3, Gd-DTPA) indicating selective delivery of these agents into the tumor. Significant Gd accumulated in the liver and kidney probably reflecting clearance and metabolism of the Gd complexes. This shows that porphyrin based agents have high potential for use as contrast agents for the detection of colorectal cells.
The difference between this data and previous animal experiments (Wedeking et at., 1992) may arise in differences in the pulse sequences, dose of Gd used, the type of cancer cells and possibly the method of measuring tissue concentration of Gd.
CONCLUSION
Methods using MR imaging and porphyrin based agents could have offered the advantage of tissue contrast enhancement and precise anatomic localization of the tumors. The porphyrin agents showed significant enhancement effect in mice with colorectal cancer cell. Further developments in MR imaging contrast agents, in combination with improved imaging techniques, may lead to novel applications in the diagnostic MR imaging. Further investigations will include using other paramagnetic ions bond to porphyrins as tumor detection agents.
Overall, with the satisfactory low levels of Gd in the liver and kidney and good tumor uptake, porphyrin based agents have considerable promise for further diagnostic applications of MR imaging. The outcome of this study may help the design of tumor-specific contrast and chemotherapeutic agents. The permeability of Gd-H and Gd-TCP, may provide tissue uptake information for both contrast and chemotherapeutic agents. With current debate on tumor-specific and therapeutic agents for tumor diagnosis and therapy, the permeability of macromolecules to tumor tissues is an essential question that needs to be answered.