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Photochemistry and Photobiology Val. 52,No. 3, pp. 491-500, 1990 Printed in Great Britain. All rights reserved 0031-8655/90$03.00+0.00 Copyright 0 1990 Pergamon Press plc In vitro EVALUATION OF PHOTOTOXIC PROPERTIES OF FOUR STRUCTURALLY RELATED BENZOPORPHYRIN DERIVATIVES ANNAM. RICHTER,~ ELIZABETH WATERFIELD,3 ASHOKK. JAIN,' ETHAND. STERNBERG,* DAVIDDOLPHIN^ and JULIAG . LEVY'* Departments of 'Microbiology and 'Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1W5 and 'Quadra Logic Technologies, Vancouver, BC, Canada (Received 20 October 1989; accepted 15 February 1990) Abstract-Four structural analogs of benzoporphyrin derivative (BPD) have been studied and compared for photosensitizing activity in v i m . All analogs have an identical reduced tetrapyrrol porphyrin ring, and differ by the position of a cyclohexadiene ring (fused at either ring A or ring B of the porphyrin) and the presence of either two acid groups or one acid and one ester group at rings C and D of the porphyrin. Photosensitizer activity was tested with the M1 tumor cell line using an assay (the MTT assay) which detects mitochondria1 hydrogenases as a measure of cell viability. This assay was shown to be equivalent to the standard clonogenicity or ['Hlthymidine uptake assay. Comparative studies with the BPD analogs showed that the monoacid derivatives had equivalent cytotoxicity and were about five-fold more active than the diacid forms. This was the case whether the assays were performed in the presence or absence of fetal calf serum. and plants, and phthalocyanines which are synthetic molecules. A few other structurally different comPhotodynamic therapy (PDT)?, a very promising pounds have been studied (for a comprehensive experimental therapy for cancer, employs photoreview see refs. Kreimer-Birnbaum, 1989; Sternberg activatable drugs and light to destroy tumor cells. and Dolphin, 1989). All the compounds currently The efficiency of PDT depends to a large extent on being investigated for prospective use in PDT have the localization of the photosensitizer in tumor cells the following characteristics in common: they are or microvasculature and its ability to be efficiently pure compounds whose structure is well known and activated by tissue penetrating light. As wavelengths absorb light in the range of 650-800 nm. In general, between 700 and 1000 nm penetrate tissue best it is difficult to compare the results obtained in (Doiron, 1984a; Doiron et al., 1984b), a photovarious laboratories because in most instances difsensitizer for use in PDT should absorb well at ferent experimental conditions and tumor models these wavelengths. Extensive clinical studies with are used. However, some comparative studies for hematoporphyrin derivative (HpD) and Photofrins different photosensitizers have been reported (a mixture of dihematoporphyrin ethersiesters, (Evensen and Moan, 1987; Moan et al., 1987). (Dougherty, 1987; Kessel et al., 1987) have been We are currently testing a chlorin-like porphyrin, generally encouraging (Dougherty, 1986; benzoporphyrin derivative (BPD), which is comMcCaughan, 1987; Gomer, 1989) and serve to mainposed of four structural analogs following synthesis. tain interest in PDT and the search for new photoAll four analogs have an identical reduced tetrapyrsensitizers. rol porphyrin ring. They differ only by the position At present, the most widely studied compounds of a cyclohexadiene ring which may be fused either for potential use in PDT are from two main groups at ring A or B of the porphyrin (ring A or B analogs) of dyes. These are porphyrins and structurally and the presence of either two acid groups (diacids) related compounds, such as chlorins, chlorophylls or one acid and one ester group (monoacids) at and purpurins, which occur naturally in animals rings C and D of the porphyrin p i g . 1). An four analogs are hydrophobic, absorb red light at about * To whom correspondence should be addressed. 700 nm wavelength, and produce singlet oxygen t Abbreviations: BPD, benzoporphyrin derivative; BPDefficiently (Bensasson, personal communication). MA, benzoporphyrin derivative, monoacid, ring A; Despite the similarity among all four molecules, BPD-DA, benzoporphyrin derivative, diacid ring A; they differ in their light activated cytotoxicity in BPD-DB, benzoporphyrin derivative, diacid ring B; vitro and in vivo. In the present paper, we report BPD-MB, benzoporphyrin derivative, monoacid, ring B; DME, Dulbecco's Modified Eagle (medium); DMSO, the results of comparative studies in vitro on photodimethyl sulfoxide; FCS, fetal calf serum; HpD, hematosensitizing activities of the analogs of BPD, in an porphyrin derivative; LD,,, lethal dose killing 50% of the population; M'IT, 3-(4,5-dimethylthiazol-2-y1)-2,5 attempt to define at a structural level those characteristics which contribute most to photosensitizer diphenyl tetrazolium bromide; PBS, phosphate buffered activity. saline; PDT, photodynamic therapy. INTRODUCTION 495 ANNAM. RICHTER et al. 496 R iR Figure 1. Structure of benzoporphyrin derivative (BPD) ( 1 ) monoacid, ring A analog, (2) monoacid, ring B analog. R = C0,Me. Diacid analogs differ from monoacid analogs only in that they have the ester group replaced with the acid group, therefore, they have two acid groups at C and D rings of the porphyrin. MATERIALS AND METHODS Synthesis of BPD analogs. Synthesis of BPD and separation of ring A and ring B monoacid and diacid analogs have been described earlier (Richter et a l . , 1987, 1989). The following analogs were obtained and tested: BPDmonoacid, ring A (BPD-MA), ring B (BPD-MB), BPD diacid, ring A (BPD-DA) and ring B (BPD-DB). All four BPD analogs were maintained in dimethyl sulfoxide (DMSO) at a concentration of 400 pg/mf. Immediately before use they were diluted in culture medium to desired concentrations. The time between the preparation of dilution and addition to the cells in culture was kept to a minimum. The photosensitizers and cells incubated with photosensitizers were protected from light at all times except for the time of planned exposure. Because final concentrations of the analogs rarely exceeded 1 pg/me, the effective dilution of DMSO was at least 1:400. At these concentrations it has no effect on cell viability. Tumor cell lines. M1 tumor cells were obtained directly from the M1 tumor (a methylcholanthrene-induced rhabdomyosarcoma of DBAR mice) grown subcutaneously in mature, male DBARJ mice as described earlier (Richter et al., 1987). The cells, obtained by teasing apart the excised tumors, were washed and cultured in 96-well plates (lo4 cells/well) in Dulbecco’s Modified Eagle (DME) medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS, Sigma Chemical Co., St. Louis, MO). Medium was changed every 24 h and the cells were used for the cytotoxicity assays before reaching full confluency, when they were still in the logarithmic growth, usually at 72 h in culture. P815 cells (mastocytoma from DBA/2 mice), KGl (human myeloid leukemia) and K562 (human myelomonocytic leukemia), all maintained in our laboratory for many years, were cultured in the same medium as M1 cells in a 10% C 0 2 humidified incubator. All cells were used for assays when they were in logarithmic growth. Cyfofoxicifyassay. The protocols for the assay differed slightly depending on the adherent or non-adherent type of cells used. M1 cells (adherent) were tested as follows: cells grown in 96-well plates, and still in logarithmic growth, were washed with DME in order to remove serum and dead, non-adherent cells. Viability of cells at this stage (as assessed by the trypan blue exclusion method) was 90% or greater. Various concentrations of photosensitizers (as well as controls) were added to wells in quadruplicate and incubated for 1 h at 37°C in the dark. Dark controls were tested on separate plates and kept under the light but covered with aluminum foil. In all tests carried out, it was found that dark controls (with or without photosensitizer) gave essentially the same cell number/ viability values as control cells (no photosensitizer) which were exposed to light. Controls of this type were run routinely with each experiment and indicated that the light had no measurable toxic effect and that the BPD analogs, at the concentrations used, had no dark toxicity. We have observed some dark toxicity with these compounds at concentrations above 5.0 pm/me. In assays testing the effect of serum, the incubation of BPD analogs with cells was carried out in the presence of 5 , 10 or 20% FCS. Immediately after the incubation, the cells were washed with DME and exposed to light in 100 pe DME/well for 1 h (5.4 J/cm2) and then incubated further in DME-5% FCS at 37°C in the dark in a 10% C 0 2 humidified incubator for 18-24 h. At that time, the viability of cells was tested using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide] (Sigma Chemical Co., St Louis, MO), as described by Mosmann (1983). The cytotoxicity assay on suspension grown cells (PS15, KG1 and K562) has been described in detail previously (Richter et a l . , 1987). These cells were used mainly to determine the relationship between the M l T assay and [3H]thymidine incorporation and clonogenicity assays. The protocol was as follows: cells in logarithmic growth (at 1O6/me) were incubated in 12 X 75 mm tubes for 1 h with various concentrations of photosensitizers in the absence of serum. Appropriate controls (no drug and dark) were included. Following the incubation the cells were washed and exposed to fluorescent light for 1 h (5.4 J/cmZ) in 0.9 me DME in tubes inclined under the light, as described earlier (Richter et al., 1987). Immediately after the exposure to light the cells were divided between the MTT assay and either the [3H]thymidine incorporation assay or clonogenic assay. At least 3 or more separate experiments were carried out for each set of conditions. MTT assay. This assay was carried out as described by Mosmann (1983). Briefly, at 18-20 h post light irradiation 10 pf of MTT at 5 mg/me PBS was added per well in 96well plates containing cells in 100 pe DME-5% FCS and the plates were placed back in the incubator for 1-3 h, at which time the reaction was stopped with 150 pe of isopropyl alcohol containing 0.04 N hydrochloric acid (Fisher Scientific). The cells were lysed and the blue formazan crystals, produced by mitochondria1 hydrogenases in living cells from the MTT substrate, were dissolved by vigorous mixing using an octapette. Full intensity of color developed within 10-15 min and the plates were read in a Bio-Rad Model 2550 EIA reader using a 600 nm filter. The percentage of cells killed was calculated in relation to control cells incubated without photosensitizer. [3H]thymidine incorporation assay. This protocol has been described previously (Richter et al., 1987). Briefly, following treatment, cells were plated in quadruplicate in 96-well plates at 1.0-1.5 x 1O5/well in DME-10% FCS and cultured in the presence of 2 pCi [3H]thymidine/well for 24 h. The amount of radioactivity incorporated by the cells served as a measure of cell viability and the ability to proliferate. The percentage of cells killed was calculated in relation to control cells incubated with medium alone. Clonogenicity assay. The protocol was as follows: following treatment, cells were cultured in duplicate in 35 mm plastic culture dishes (LUX 5221-R, Miles Scientific, Naperville, IL) at 5 x lo4 KG1 cells/dish in 1 mf of DME medium containing 10% FCS, 1% methylcellulose (Fluka AG, CH-9470 Buchs, Switzerland) and 50 p M of 2-mercaptoethanol (Eastman Kodak Co., Rochester, NY). The dishes were incubated in a CO, incubator and colonies were counted by inverted microscope 14 days later. The percentage of proliferating cells killed was calculated by relating the number of colonies formed after treatment to the number of colonies formed by control Photosensitizing potency of benzoporphyrin derivatives cell preparations. The large inoculum reflects the low cloning efficiency of KG1 due to the presence in the cultures of large numbers of terminally differentiated cells which were no longer capable of cell division. Inoculum size was based empirically on numbers which yielded between 100 and 200 colonies per plate. The minimum number of cells defined as a colony was 12-15 cells. Light source. For light irradiation, a bank of 4 fluorescent lamps (General Electric F20T12, Delux Cool White) was used. The spectrum of light was between 300 and 800 nm with the highest radiant power at 600 nm (G.E. Bulletin, unpublished data). The incident light density at 11 cm distance from the lamp (routine distance for the light irradiation of plates and tubes) was 1.5 mWI cmz as measured by YSI Kettering Model 65 radiometer (Yellow Springs, OH). This instrument is a wide spectrum radiometer with greatest sensitivity at about 700 nm. The temperature at this distance did not exceed 25°C at any time. RESULTS Characteristics of BPD analogs The structures of BPD analogs are shown in Fig. 1. The molecular weights for diacids and monoacids are 704 and 718, respectively. They are lipophi!ic molecules with limited solubility in aqueous solutions. All four analogs have very similar absorption spectra (Fig. 2) and absorb light efficiently in the red part of the spectrum. The extinction coefficient in dichloromethane is 34 000 M-lcm-' for BPDMA and within 25% of this value for other analogs. In aqueous solutions the 688 nm absorption peak shifts to 692 nm. Further shift to the right is indicative of aggregation and increases with the concentration of BPD and with time in an aqueous solution. For this reason, comparative absorption spectra were consistently measured in 50% methanol. All four analogs emit red fluorescence i --+--.+--+-d----d 400 500 600 700 800 497 (690 nm) when excited at 420 nm. Efficient excitation can also be achieved within the UV range (355 nm; Jamieson et al., 1989). Assays for determination of cell survival after photodynamic treatment In order to compare the 4 BPD analogs' ability to kill test cell lines, we have developed an assay system which measures viability colorimetrically by measuring the activity of mitochondria1 hydrogenases. The assay has been described previously (Mosmann, 1983) and the concentration of blue formazan, produced by cleavage of the yellow tetrazolium salt (MTT) by hydrogenases in mitochondria of living cells, has been shown to correlate both with the number of viable cells and their metabolic activity, including the ability to proliferate. This test has been adapted in our laboratory for its speed and convenience which enabled us to screen many photosensitizers under diverse conditions. Incubation of cells after treatment, for about 24 h prior to the MTT assay, was selected in order to standardize the assay and has been determined experimentally. Preliminary studies, in which the M'TT assay was compared to trypan blue exclusion as a measure of cell viability following photosensitization of cells, indicated good correlation. We have also confirmed the correlation, reported by Mosmann (1983), between the MTT assay and ["Hlthymidine incorporation using various cell lines (Fig. 3). In the present study, we also compared the MITT assay to a clonogenic assay using the KG1 cell line and various doses of the BPD analog, BPD-MA. In these experiments cells were exposed to BPD-MA in the absence of serum. Following light exposure, cells were split and incubated for 24 h in DME-5% FCS (MTT assay) or cultured for 14 days in the 100 1 0 2 0 4 0 60 8 0 100 3H-THYMIDINE ASSAY WAVELENGTH (nrn) Figure 2. Absorption spectrum of BPD-MA (15 pg/mt) in 50% methanol-PBS solvent. The peaks, indicated by arrows, are at 354, 418, 574, 626 and 688 nm wavelength. All 4 BPD analogs have almost identical spectra (not shown for clarity) and very close extinction coefficients (within 25% range). Figure 3. Correlation between the results of MTT assay and [3H]thymidine incorporation assay. The percentage of cells (P815, KG1, K562 cell lines) killed after the exposure to the photosensitizer (BPD-DA or -DB) and light (5.4JI cmz) was determined parallely by MTT assay (20-23 h post exposure) and [3H]thymidine incorporation (0-24 h post exposure). ANNAM. RICHTER et a1 498 presence of 10% FCS under the conditions described in the Materials and Methods section. The results (Fig. 4) show that the MTT assay correlates well with the clonogenic assay in determining cell survival. Similar results were obtained when MTT and clonogenicity were compared with the P815 cell line (data not shown). Cytotoxicity of BPD analogs In the cytotoxicity assay developed, we routinely use serum-free conditions, because serum might complicate the in vitro system, without actually duplicating the in vivo situation. The presence of serum elements could modify drug-cell interaction in vitro without mimicking the in vivo situation. However, we have compared the 4 analogs of BPD both in the absence and in the presence of 5-20% FCS. The photosensitizing activity of the 4 BPD analogs tested using M1 cells in serum-free medium differed, especially between monoacid and diacid analogs, monoacids being at least 5 times more potent than diacids (Fig. 5). Clearly, the presence of one or two acid groups on rings C or D of the porphyrin macrocycle has a major effect on the photosensitizing activity of this family of molecules. The concentrations of BPD-MA, -MA, -DA, and -DB required to kill 50% of cells (LD50) in the absence of serum were 21, 19, 75, and 105 ng/me culture medium, respectively. The presence of FCS during the incubation with BPD analogs did not change the order of their photosensitizing activity (BPD-MA > = -MB -DA > -DB), merely increasing the concentration of the analogs required to achieve the same photodynamic killing (Fig. 6). The LDSo of BPD analogs in the presence of serum ~. were proportional to the concentration of serum, roughly doubling with doubling serum concentrations. * DISCUSSION The physical properties of the BPD analogs studied here do not differ significantly, nor does their ability to produce singlet oxygen (Dr. Bensasson, personal communication). The present study was undertaken in an attempt to determine whether any of the features which distinguish the analogs from each other would contribute to greater phototoxicity of malignant cells in vitro. During the past two years, we have utilized a rapid assay system which appears to correlate well with clonogenic assays. The MTT test system involves the colorimetric detection of active mitochondrial hydrogenases as a measure of cell viability. The assay is rapid and easy to perform and permits a reliable procedure for screening photosensitizer activity. The MTT assay has been reported by others to be useful in determining the effects of chemotherapeutic drugs on human leukemia (Campling et al., 1988; Pieters et al., 1988) and lung cells (Cole, 1986). We also found that the most reliable procedure for establishing values for phototoxic killing involved the incubation of treated cells overnight before determining rates of killing. In the study reported here, we have shown that this procedure, using the M'IT assay, correlates closely with more standard procedures involving [)H]thymidine incorporation (Fig. 3) and estimates of clonogenicity (Fig. 4), both of which also determine viability of cells following the time required for phototoxic killing. McHale and McHale (1988) have also shown the correlation between the M7T and the standard clonogenic assay while determining human breast and melanoma cell survival following 1001 100 - 80 - ) . > 20 . a v fn 6 0 - a . t I- s 40- : 0 2 0 4 0 60 80 100 CLONOGENIC ASSAY Figure 4. Correlation between M'IT and clonogenic assays. The percentage of KGl cells killed by exposure to various concentrations of BPD-MA and light (5.4 Jim2) was determined parallely by MTT and clonogenic assays. Error bars indicate standard error. - - BPD-DB Photosensitizing potency of benzoporphyrin derivatives 100- (A) 8060 - 40 - 100 0 % FCS 5 % FCS 10 % FCS 20 % FCS 10' 102 1 o3 Log. Conc. (ng/ml) Log. Conc. (nglml) Figure 6. Photosensitizing potency of BPD-MA (A) and BPD-DA (B) toward M1 tumor cells in culture. The cells were incubated with either of the photosensitizers in the absence of serum or in the presence of 5, 10, or 20% of serum for 1 h and then washed and exposed to light (5.4 J/ cm'). The percentage of cells killed was determined by MTT assay at 18-24 h post exposure. Each point represents the average of 4-6 experiments. Error bars indicate the standard error. photoradiation therapy with HpD. Good correlation between the MTT assay and ["Hluridine incorporation in human tumor cell lines after treatment with doxorubicin or vindesine has also been reported (Ford et al., 1988). We have carried out many experiments in the absence of serum. Our reasoning for performing experiments in this way was based on our wish to evaluate these compounds directly, in terms of their action on cells, in the absence of any possible modifying effects introduced by serum proteins. Not surprisingly, serum components were found to compete with the cells for photosensitizer; therefore, more photosensitizer molecules were required for the same photodynamic damage, while the mechanism of photosensitization (as judged from the paralleled slopes, Fig. 6) is the same. In the presence of serum, the hierarchy of efficacy between BPD analogs established in the absence of serum remained unchanged. We have shown here using the 4 BPD analogs that relatively minor structural differences may affect the photosensitizing activity of molecules. It was evident that the position of the cyclohexadiene ring at the A or B ring of porphyrin does not affect the photosensitizing efficiency of the isomers as much as the presence of two acids or one acid and one ester group at rings C and D of the porphyrin macrocycle. However, the presence of the cyclohexadiene ring 499 fused to the porphyrin macrocycle may be responsible for relatively high photosensitizing potency of BPD analogs. Morgan et al., (1987) have found that photosensitizing potency was related to the presence of "bulky substitution" in the form of a five or six member ring at the reduced porphyrin macrocycle. The major difference between mono and diacid forms of BPD is their relative hydrophobicity and lipophilicity. Although all BPD analogs have very limited solubility in aqueous solutions, monoacids are more soluble than diacids. Limited water solubility has been reported to make disulfonated zinc phthalocyanine more efficient as a photosensitizer than other forms, either insoluble or more soluble (Brasseur et al., 1988). Also, BPD monoacids are more lipophilic than the diacid forms. It is possible that this feature could enhance their association and uptake by cell membranes. It was reported earlier by Kessel (1989) that BPD-MA was more readily taken up by L1210 cells than was BPD-DA. BPD diacids are more negatively charged than monoacid forms in neutral aqueous solutions (or in serum). It is not possible to speculate at this time as to whether or not the net negative charge on cell surfaces influences relative uptake of the various BPD analogs. Cellular uptake and localization of the BPD analogs is currently under investigation. Acknowledgements-The authors express their gratitude to Ms. Kathy Lamb and Mr. Stephen Yip for their skillful technical assistance. Research supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada, Grant No. 5-80268. REFERENCES Brasseur, N., H. Ali, R. Langlois and J. E. van Lier (1988) Biological activities of phthalocyanines-IX. Photosensitization of V-79 Chinese hamster cells and EMT-6 mouse mammary tumor by selectively sulfonated zinc phthalocyanines Photochem. Photobiol. 47, 705-71 1. Campling, B. G . , J. Pym, P. R . Galbraith and S. P. C. Cole (1988) Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leuk. Res. 12, 823-831. Cole, S. P. C. (1986) Rapid chemosensitivity testing of human lung tumor cells using the MTI' assay. Cancer Chemother. Pharmacol. 17, 259-263. Doiron D. R. (1984a) Photophysics and instrumentation for porphyrin detection and activation. In Porphyrin Localization und Treatment of Tumors (Edited by D . R. Doiron and C. J. Gomer), p.41. Liss, New York. Doiron, D. R., C. J. Gomer, S. W. Fountain and N. J . Razum (1984b) Photophysics and dosimetry of photoradiation therapy. In Porphyrins in Tumor Phototherapy (Edited by A. Andreoni and R. Cubeddu), pp. 281-291. Plenum Press, New York. Dougherty, T. J. (1986) Photosensitization of malignant tumors. Semin. Surg. Oncol. 2, 24-37. Dougherty, T. J. (1987) Studies on the structure of porphyrins contained in Photofrin 11. Photochem. Photobiol. 46, 569-573. Evensen, J . F. and J . Moan (1987) A test of different photosensitizers for photodynamic treatment of cancer in a murine tumor model. Photochem. Photobiol. 46, 859-865.