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Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing.

Abstract

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 [mm.sup.3] the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/[cm.sup.2], 168 J/[cm.sup.2]).

All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05).

Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Hypericin; Cancerotherapy; Photodynamic therapy; Tumour

Introduction

Photodynamic therapy (PDT) is an established mode of treatment for various diseases involving cell hyperproliferation, including cancer. The treatment typically involves systemic administration of a tumour-localizing photosensitizer and its subsequent activation by light of an appropriate wavelength to create a photochemical reaction causing photodamage to the tumour (Dougherty et al., 1998).

Hypericin is a plant pigment isolated from Hypericum perforatum L. which displays several photodynamic actions (Diwu, 1995). The photodynamic properties of hypericin have been tested in vitro and in vivo by several experimental studies (VanderWerf, 1996; Vandenbogaerde, 1998; Cavarga et al., 2001; Solar et al., 2002).

Three mechanisms of PDT-mediated tumour destruction in vivo have been investigated most intensively. The first mechanism is direct tumour cell killing, the second involves damaging the tumour-associated vasculature with subsequent ischemic necrosis, and the third mechanism is post-treatment immune response against cancer cells (Dougherty et al., 1998; Dolmans et al., 2002). In recent years the importance of direct photodynamic damage to the tumour vasculature has been stressed as an effective strategy in therapy against solid tumours. Targeting the tumour vasculature by applying a short interval between drug administration and photoirradiation (short drug-light interval) in PDT with hypericin is more effective than a longer drug-light interval allowing the tumoural concentration of hypericin to peak (Chen et al., 2001; Chen et al., 2002). However, most current clinical PDT protocols use a drug-light interval with the maximal concentration ratio between the tumour and its surrounding healthy tissue (Dougherty et al., 1998). Dolmans et al. (2002) demonstrated in their in vivo study that PDT involving multiple administration of pyropheophorbide photosensitizer at appropriate times prior to a single activating light dose, targets both the vascular and tissue compartments of the tumour. This approach was more effective than a single administration of the drug.

To our knowledge experimental results of fractionated systemic dosage of hypericin in vivo have so far not been published. In this study it was decided to use intraperitoneal single and fractionated schedules of administration of hypericin, and photodynamic therapy efficacy was subsequently evaluated. Experiments were performed on the murine fibrosarcoma model.

Materials and methods

Photosensitizer

Hypericin was purchased from Molecular Probes, Eugene, Oregon, USA. Hypericin was dissolved in a mixture of ethanol and glycerol (1:1, vol/vol). Then a solution of 20% PEG 400 in PBS (vol/vol) was added to the final concentration of hypericin 0.1% (wt/vol). Hypericin was prepared in subdued light conditions. The drug was used immediately after preparation.

Animals

Male C3H/DiSn inbred mice, 8-10 weeks old (weighing ~20 g), were obtained from VELAZ, Prague, Czech Republic. The animals were quarantined for a period of 2 weeks and were housed in rodent cages with 4-5 animals per cage at about 23[degrees]C, and they were given Velaz/Altromin 1320 St lab chow and tap water acidified to pH 2.4 ad libitum. During therapy experiments, the mice were housed in cages under subdued light conditions. The research was conducted according to the principles enunciated in the "Guide for the Care and Use of Laboratory Animals", prepared by the State Veterinary Office of the Slovak Republic.

Cell line and tumour response studies

The N-methyl-N'-nitro-N-nitrosoguanidine-induced G:5:1:13 fibrosarcoma cell line (H-[2.sup.k]) was kindly provided by Dr. Margaret L. Kripke (University of Texas, M. D. Anderson Cancer Center, Houston, Texas, USA). G:5:1:13 cells were maintained in RPMI-1640 culture medium at 37[degrees]C with 5% C[O.sub.2] atmosphere and were used for experiments during the exponential growth phase. Tumour cells were harvested by trypsinization, washed twice with serum-free medium and adjusted to the concentration of 8 X [10.sup.5] cells/ml. Anaesthetised animals were injected with 1 X [10.sup.5] viable tumour cells/mouse s.c. into the depilated paramedian lumbar region. Incidence and approximate tumour size were recorded weekly throughout the experimental period. Tumour location was determined by palpation. Tumour size was determined up to 91 days by measuring three dimensions of each tumour using a calliper. Tumour volumes ([mm.sup.3]) were calculated using the formula for a hemiellipsoid (V = [pi]/6 X L X W X H; L, W and H designate tumour diameters for length, width and height, respectively). Results are presented as Kaplan-Meier curves in which the percentage of animals whose tumour volume increases less than five-fold is plotted against the number of days post-treatment.

Survival was monitored daily and the dying animals in this experiment were euthanized by cervical dislocation when moribund.

Light delivery

Photodynamic therapy was performed using light provided by solid-state laser. The excitation wavelength used in the experiments was 532 nm. The light dose of 168 J/[cm.sup.2] was delivered at the fluence rate 70 mW/[cm.sup.2]. The irradiation spot centred on the tumour region was 1 cm in diameter.

Photodynamic therapy protocol

Mice bearing G:5:1:13 tumours were randomised into six groups of 5-9 animals per group for the following treatments:

(1) No treatment (9);

(2) Laser light (5);

(3) Hypericin alone i.p. (5 mg/kg) (5);

(4) Single dose of hypericin i.p. 1 h before laser irradiation (5 mg/kg) (7);

(5) Single dose of hypericin i.p. 6 h before laser irradiation (5 mg/kg) (6);

(6) Fractionated dose of hypericin i.p. 2.5 mg/kg 6 h and 2.5 mg/kg 1 h before laser irradiation (6).

Mice were treated with hypericin when the tumour reached a volume of 40-80 [mm.sup.3] (~17 days after inoculation). Hypericin was administered intraperitoneally at a volume 200 [micro]l of solution. With delays of 1 or 6 h after application in treatment groups 4-6, tumours were irradiated with laser light. Immediately prior to irradiation the mice were anaesthetised with Narkamon/Rometar. In order to avoid photosensitizer activation by ambient light, animals were kept in covered cages in subdued light conditions after hypericin administration.

Statistics

Differences in survival between treated and control mice were assessed using the [chi square]-test with Yates' correction. The significance of differences between respective groups was analysed by nonparametric techniques (Kruskal-Wallis ANOVA and Mann-Whitney test). All statistical calculations were done using Arcus QuickStat Biomedical ver.1.1.

Results

The growth curves for untreated tumours and tumours treated with hypericin or laser alone were almost identical with those calculated for G:5:1:13 fibrosarcoma in our previous study (Cavarga et al., 2001). All tumours in these control groups exceeded the initial volume five-fold between the 21st and 28th day of the experiment (Fig. 1). None of these tumours regressed spontaneously and none of animals survived the 70th day of experiment (Fig. 2). The median survival time of tumour-bearing mice was 59 days (range 55-64 days) for non-treated control, 50 days (range 47-60 days) for the group irradiated by laser and 60 days (range 49-64 days) for the group treated with hypericin alone.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

A thick necrotic eschar was apparent in PDT-treated groups of animals 48h after laser light exposure. Necrosis of the tumoural area persisted for several days. Complete remission (CR) of the tumour was observed in one of seven animals in the hypericin-PDT-treated group with single drug dose (5 mg/kg) administered 1 h before irradiation. A similar result (one of six animals) was obtained at the 6 h drug-light application interval. Long-term survival was registered in these PDT-treated groups only in animals with CR of G:5:1:13 fibrosarcomas. On the other hand, two of six animals had complete remission of tumour (Fig. 1) and one mouse survived with tumour growth inhibition in the group with fractionated administration of hypericin 6 and 1 h before irradiation. The median survival time of tumour-bearing mice was prolonged from 47 days (range 36-58 days) in the group of animals with a single drug dose administered 6 h before irradiation to 64 days (range 55-71 days) for the group with 1 h drug-light interval, and to 84 days (range 69-91 + days) in the group with fractionated administration of hypericin 6 and 1 h before irradiation. This PDT protocol increased the survival rate of animals up to the 91st day of observation with statistical significance (p < 0.05) as compared with single dose schedules (Fig. 2).

Discussion

Most clinical strategies for cancer therapy aim at direct killing of the malignant tumour cells. An alternative approach to tumour therapy is to induce cell necrosis by occlusion of the tumour blood supply. Chen et al. (2001) found that hypericin was exclusively located in the vascular network of the tumour 30 min after i.v. injection. The compound was located predominantly in interstitial and cellular compartments of the tumour 6 and 24 h after i.v. administration. PDT at 30 min drug-light interval produced 100% cure of RIF-1 murine tumours. The percentage of cures decreased very rapidly as a function of the interval between drug administration and light irradiation (Chen et al., 2002). However, in our study 1 h after i.p. application the G5:1:13 fibrosarcoma completely regressed in only one mouse out of seven (i.e. 14% of treated animals). A similar result was obtained at 6 h drug-light administration interval (CR in one of six, i.e 17% of treated mice). Although our results differ from these of Chen et al. (2002), similar observations to ours were presented by Du et al. (2003), who also observed no significant differences in hypericin-based PDT at 1 and 6 h administration intervals. Dolmans et al. (2002) used an interesting approach. They performed single and fractionated dosing of the pyropheophorbide photosensitizer in the murine mammary adenocarcinoma model. The photosensitizer was applied in single doses i.v. 15 min and 4 h before laser irradiation. When the total drug dose of 0.03 mg/kg body weight was fractionated into equal drug doses and the fractions were administered at 4 h and 15 min before the light exposure, a significant tumour growth delay was observed compared with single full drug dosing at either 4 h or 15 min before light exposure. A fractionated hypericin dosing schedule was also used in our experiment. Intraperitoneal doses 2.5 mg/kg at 6 h and 2.5 mg/kg at 1 h before laser irradiation was more efficient than a single dose 5 mg/kg at either 6 or 1 h before laser application, as measured by survival assay. There are several explanations for these observations. Both luminal and abluminal surfaces of the vascular wall are targeted in fractionated photosensitizer application, and thus PDT may more effectively damage tumour vasculature. Secondly, tumour blood flow is known to be temporally and spatially heterogeneous. This temporal and spatial heterogeneity in permeability may hinder the delivery of therapeutic agents in a single dose. Fractionated drug application can provide better tissue distribution of the photosensitizer (Monsky et al., 1999; Dolmans et al., 2002).

In summary: our experimental model shows that fractionated administration of hypericin can produce better therapeutic response than single administration.

Acknowledgements

This work was supported by Science and Technology Assistance Agency under the contract No. APVT-20-022202, grant No. 1/9211/02 of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and Institutional grant of Faculty of Medicine, P. J. Safarik University No. 8/2002/IG4.

References

Cavarga, I., Brezani, P., Cekanova-Figurova, M., Solar, P., Fedorocko, P., Miskovsky, P., 2001. Photodynamic therapy of murine fibrosarcoma with topical and systemic administration of hypericin. Phytomedicine 8, 325-330.

Chen, B., Xu, Y., Roskams, T., Delaey, E., Agostinis, P., Vandenheede, J.R., De Witte, P., 2001. Efficacy of antitumoral photodynamic therapy with hypericin: relationship between biodistribution and photodynamic effect in the RIF-1 mouse tumor model. Int. J. Cancer 93, 275-282.

Chen, B., Roskams, T., De Witte, P., 2002. Antivascular tumor eradication by hypericin-mediated photodynamic therapy. J. Photochem. Photobiol. 76, 509-513.

Diwu, Z., 1995. Novel therapeutic and diagnostic application of hypocrellins and hypericins. J. Photochem. Photobiol. 61, 529-539.

Dolmans, D.E.G.J., Kadambi, A., Hill, J.S., Flores, K.R., Gerber, J.N., Walker, J.P., Borel Rinkes, I.H.M., Jain, R.K., Fukumura, D., 2002. Targeting tumor vasculature and cancer cells in orthotopic breast tumor by fractionated photosensitizer dosing photodynamic therapy. Cancer Res. 62, 4289-4294.

Dougherty, T.J., Gomer, C.J., Henderson, B.W., Jori, G., Kessel, D., Korbelik, M., Moan, J., Peng, Q., 1998. Photodynamic Therapy. J. Natl. Cancer Inst. 90, 889-905.

Du, H.Y., Bay, B.H., Olivo, M., 2003. Biodistribution and photodynamic therapy with hypericin in human NPC murine tumor model. Int. J. Oncol. 22, 1019-1024.

Monsky, W.L., Fukumura, D., Gohongi, T., Ancukiewicz, M., Weich, H., Torchilin, V.P., Yuan, F., Jain, R.K., 1999. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endotelial growth factor. Cancer Res. 59, 4129-4135.

Solar, P., Cavarga, I., Hofmanova, J., Cekanova-Figurova, M., Miskovsky, P., Brezani, P., Hrckova, G., Kozubik, A., Fedorocko, P., 2002. Effect of acetazolamide on hypericin photocytotoxicity. Planta Med. 68, 658-660.

Vandenbogaerde, A.L., Delaey, E.M., Vantieghem, A.M., Himpens, B.E., Merlevede, W.J., De Witte, P., 1998. Cytotoxicity and antiproliferative effect of hypericin and derivatives after photosensitisation. J. Photochem. Photobiol. 67, 119-125.

VanderWerf, Q.M., Saxton, R.E., Chang, A., Horton, D., Paiva, M., Anderson, J., Foote, C.H., Soudant, J., Mathey, A., Castro, D.J., 1996. Hypericin: a new laser phototargeting agent for human cancer cells. Laryngoscope 106, 479-483.

I. Cavarga (a), P. Brezani (b), P. Fedorocko (c), P. Miskovsky (c,d,*), N. Bobrov (b), F. Longauer (b), S. Rybarova (b), L. Mirossay (b), J. Stubna (b)

(a) Medical School Hospital of L. Pasteur, Kosice, Slovakia

(b) Faculty of Medicine. P. J. Safarik University, Kosice, Slovakia

(c) Faculty of Sciences, P. J. Safarik University Kosice, Slovakia

(d) International Laser Center, Bratislava, Slovakia

Received 26 January 2004; accepted 20 February 2004

*Corresponding author. Faculty of Sciences, P. J. Safarik University Kosice, Slovakia. Tel.: + 55 6222986; fax: +55 6222124.

E-mail address: misko@kosice.upjs.sk (P. Miskovsky).
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Title Annotation:SHORT COMMUNICATION
Author:Cavarga, I.; Brezani, P.; Fedorocko, P.; Miskovsky, P.; Bobrov, N.; Longauer, F.; Rybarova, S.; Miro
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Sep 1, 2005
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