Van de Putte 5_9

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. Introduction Hypericin, a polycyclic aromatic naphthodianthrone constituent (Fig. 1) of the plant genus Hypericum, is a potent photosensitizer for photodynamic therapy (PDT) of tumors. Hypericin has proven to selectively accumulate in tumor tissue and to induce a pronounced antitumoral effect upon light irradiation (1,2). After intraperitoneal administration of 5 mg/kg hypericin, a 16-fold higher concentration of hypericin in tumor tissue versus surrounding healthy tissue was found in a subcutaneous P388 lymphoma tumor (3). Similar encouraging results were found with RIF-1 fibrosarcoma tumors (4). Moreover, a study performed with radioactive 123I-labeled hypericin showed that the selective tumor accumulation is not dependent on the amount administered, as the maximal retention of radioactivity in tumor tissue was similar for the carrier-added and no-carrier added conditions (5). Hypericin, which is almost insoluble in water, disperses in a physiological aqueous environment, producing nonfluorescent high molecular weight aggregates (6). Stability studies previously performed in our laboratory demonstrated that both the formulation of hypericin concentrates and the storage temperature have a substantial impact on the formation of aggregates. It was shown that polar organic solvents (e.g., polyethylene glycol) form stable fluorescent solutions, whereas aqueous vehicles were associated with a loss in photoactive content during storage and only partially released their hypericin content when applied to cells (7). Of importance, hypericin aggregates can also be formed during synthetic work, i.e. when the time of irradiation necessary to convert proto-hypericin via an oxidative photocyclization reaction into hypericin, is inadequately long. Despite the fact that in the presence of lipoproteins bloodborne hypericin aggregates can readily dissociate into the monomeric form (8,9), we noticed in the course of a preliminary study that i.v. administration of aggregated compound strongly hindered a selective accumulation in tumor tissue. In this work we examined quantitatively the impact of aggregates present in the delivery mixture on the biodistribution of hypericin. For that purpose, 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. To support INTERNATIONAL JOURNAL OF ONCOLOGY 28: 655-660, 2006 655 The impact of aggregation on the biodistribution of hypericin MARIE VAN DE PUTTE1, TANIA ROSKAMS2, GUY BORMANS3, ALFONS VERBRUGGEN3 and PETER A.M. DE WITTE1 1Laboratorium voor Farmaceutische Biologie en Fytofarmacologie, Faculteit Farmaceutische Wetenschappen; 2Afdeling Histochemie en Cytochemie, Faculteit Geneeskunde; 3Laboratorium voor Radiofarmaceutische Chemie, Faculteit Farmaceutische Wetenschappen, K.U. Leuven, Belgium Received September 5, 2005; Accepted October 21, 2005 _________________________________________ Correspondence to: Dr Peter A.M. De Witte, Laboratorium voor Farmaceutische Biologie, Faculteit Farmaceutische Wetenschappen, K.U. Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium E-mail: [email protected]