Hypericin is a potent agent in the photodynamic therapy of cancers and accumulates to a large extent in tumor tissue. To better understand the impact of hypericin aggregates present in the delivery vehicle on the biodistribution of the compound, we compared the in vivo tissue accumulation after administering hypericin suspended as coarse aggregates in phosphate-buffered saline, with the biodistribution found after injection of a solution of hypericin in a mixture of DMSO, polyethylene glycol and water. When administered as coarse aggregates, hypericin showed a pronounced uptake in liver, spleen and lung and a slow body clearance with a complete decline in tumor/normal tissue ratios (far less than 1). In contrast, delivery of hypericin as a solution resulted in dramatically improved tumor to normal tissue ratios and a relatively fast elimination from the body. To elucidate the exact localization of hypericin in both conditions, a fluorescence microscopy study was performed on sections of spleen, liver, lung and tumor tissue. At 24 h after injection, fluorescence in spleen, liver and lung was faint and homogeneous for dissolved hypericin, whereas bright fluorescent spots covering the entire tissue sections were found when coarse aggregates were injected. We found that aggregates get trapped within these tissues, followed by a gradual monomerization. A direct involvement of monocytes and macrophages, however, could not be demonstrated. In conclusion, it is of critical importance that the delivery vehicle prevents extensive aggregation of hypericin before injection and assures an efficient transfer to serum lipoproteins upon injection. These results may also be extended to radiolabeled derivatives and other lipophilic photosensitizers, such as porphyrins, phthalocyanines, naphthalocyanines and chlorines, with similar aggregation properties.

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In this proof-of-principle study, the necrosis avid agent hypericin was investigated as a potential indicator for early therapeutic response following radiofrequency ablation (RFA) of murine liver tumors. Eight mice bearing intrahepatic RIF-1 tumors were intravenously injected with hypericin 1 h before or 24 h after RFA treatment. Mice were euthanized 24 h after hypericin injection and excised livers were investigated by means of fluoromacroscopic and fluoromicroscopic examinations in combination with conventional histomorphology. Significant differences in hypericin fluorescence were found in necrosis, viable tumor and normal liver tissue in a decreasing order: in necrosis, mean fluorescence densities were about 5 times higher than in viable tumor and approximately 12 times higher than in normal liver (p<0.05). Mean fluorescence densities were not significantly different when hypericin was injected 24 h after or 1 h before RFA treatment (p>0.05). As a conclusion, hypericin features the property to specifically enhance the imaging contrast between necrotic and viable tissues and to non-specifically distinguish viable tumor from normal liver. The results suggest that hypericin offers significant potential in the early assessment of response following necrosis-inducing antineoplastic treatments such as RFA.

In this study we have compared the tumour-seeking properties of mono-[(123)I]iodoprotohypericin and mono-[(123)I]iodohypericin in C3H mice with a subcutaneous radiation-induced fibrosarcoma-1 tumor. After intravenous injection, both tracers were rapidly cleared from all organs and were retained by the tumors. There was no significant difference in tumor uptake of the two tracers at all studied time points (p > 0.05). To study the plausible mechanism of hypericin and mono-iodohypericin uptake in tumor, their plasma binding profile was investigated. Both agents show high affinity for low-density lipoproteins and to a lesser extent high-density lipoproteins and other heavy proteins. Mono-[(123)I]iodohypericin appears to be more promising as a tumor diagnostic agent, given its faster clearance from all organs.

It is estimated that 30–80% of solid tumor mass represents necrotic tissue that consists out of a significant number of dead and dying cells. The fact that these necrotic zones are restricted to dysplastic and malignant tissue and are rarely present in normal tissue makes necrosis an interesting target both for cancer diagnosis and therapy. In this study, the avidity of hypericin, [123I]iodohypericin and [131I]iodohypericin to tumor necrosis was explored for both diagnosis and therapy of experimental malignancies. The intratumoral distribution in RIF‐1 tumors was investigated by means of fluorescence microscopy (hypericin) and autoradiography ([123I]iodohypericin). Results show high uptake of the tracers in necrosis at 24 hr, lasting for up to 72 hr p.i. Ratios of activity of [123I]iodohypericin in necrotic tissue over viable tumor reached up to 19.63 ± 4.66, correlating with 9.20% ID/g in necrosis. Nude mice bearing RIF‐1 tumors that received three injections of 300 μCi over a 3‐week treatment period showed stabilization in tumor growth for 5 days, as measured by caliper and micro‐positron emission tomography using [18F]fluorodeoxyglucose. Based on these results, we suggest the potentials of radiolabeled hypericin (1) in diagnostic aspects including prognosis or staging assessment of bulky necrotic cancers, monitoring of treatments and therapeutic follow‐up; and (2) in cancer treatment based on tumor necrosis. In conclusion, we showed that hypericin radiolabeled with iodine is a necrosis avid tracer that can be used both as a tumor diagnostic and therapeutic.