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Photochemistry and Photobiology Val. 52, No. 3. pp. 501-507, 1990 Printed in Great Britain. All rights reserved

Copyright 0 1990 Pergamon Press plc

THE PLASMA DISTRIBUTION OF BENZOPORPHYRIN DERIVATIVE AND THE EFFECTS OF PLASMA LIPOPROTEINS ON ITS BIODISTRIBUTION BETHA. ALLISON,'*P. HAYDN PRITCHARD,~ ANNAM. RICHTER] and JULIAG. L E W ~ Departments of 'Microbiology and ZPathology, University of British Columbia, Vancouver, British Columbia. Canada V6T 1W5

(Received 25 October 1989; accepted 21 February 1990) Abstract-The plasma distribution and biodistribution of benzoporphyrin derivative were examined. Two analogs of benzoporphyrin derivative were mixed with human plasma in vitro and recovered in the lipoprotein fractions upon separation by chromatography or ultracentrifugation. The majority of both analogs was recovered with high density lipoprotein. The effect of prebinding benzoporphyrin derivative to lipoproteins on the biodistribution of the drug in vivo was studied in tumor bearing DBN2.I mice. At 3, 8 and 24 h post-injection, tumor and tissue samples were excised and analyzed for benzoporphyrin derivative content. Precomplexing benzoporphyrin derivative with low density lipoprotein or high density lipoprotein led to significantly (P < 0.05) greater tumor accumulation than in aqueous solution. INTRODUCTION

We have recently described the cytotoxicity (Richter et al., 1987) and biodistribution (Richter et a l . , 1989a) of a potent new photosensitizer, benzoporphyrin derivative (BPD)?. This compound absorbs light strongly at 692 nm and is composed of four structural analogs following synthesis. Biodistribution studies show that BPD-monoacid, ring A (BPD-MA) achieves a favorable tumor : normal tissue ratio within 3 h and is a good candidate for photodynamic therapy (PDT). The factors affecting the biodistribution of BPD are poorly understood. However, there have been several reports in the literature which implicate plasma lipoproteins in the binding and transport of porphyrins and other photosensitizers. For example, hematoporphyrin (Hp) (Jori et a l . , 1984), protoand uroporphyrin (Reyftmann et a l . , 1984) and hematoperphyrin derivative (Kessel, 1986) have all been reported to bind to plasma lipoproteins. As a result, much interest has been generated regarding the influence of lipoproteins on delivery of porphyrins to abnormal tissue. Barel et al. (1986) observed that formation of complexes of Hp and low density lipoprotein (LDL) led to more specific delivery to tumor tissue. In addition, Zhou et al. (1988) reported that the association of Hp with LDL may speed up direct damage to neoplastic cells in response to PDT. *To whom correspondence should be addressed. TAbbreviations: BPD, benzoporphyrin derivative; BPDDA, benzoporphyrin derivative diacid ring A; BPDMA, benzoporphyrin derivative monoacid ring A; DMSO, dimethyl sulfoxide; HDL, high density lipoprotein; Hp, hematoporphyrin; LDL, low density lipoprotein; PDT, photodynamic therapy; PBS, phosphate buffered saline; TLC, thin layer chromatography; VLDL, very low density lipoprotein.

Preliminary studies by Kessel (1989) on the association of BPD-MA and BPD-diacid, ring A (BPD-DA) with plasma lipoproteins indicated that these analogs of BPD bound primarily to the high density lipoprotein (HDL) fraction, although significant amounts also associated with LDL. In the present study, the plasma distribution of BPD-MA and BPD-DA was examined and the contribution of albumin binding to the BPD recovered in the HDL fraction was found to be negligible. Therefore, we have investigated the effect of precomplexing BPD-MA with purified lipoproteins on the subsequent biodistribution in vivo. The results indicate that prebinding BPD-MA with HDL or LDL leads to significantly enhanced tumor deposition. MATERIALS AND METHODS

Synthesis ofphotosensitizers. Synthesis of BPD (Richter et al., 1987) results in four structural analogs (Fig. 1). The two analogs used in this study differed only by the presence of either two acid groups (diacid, BPD-DA) or one acid and one ester group (monoacid, BPD-MA). In these analogs the cyclohexadiene ring was fused on ring A of the porphyrin. The ratio between mono and diacids formed depends upon the length of hydrolysis with 25% hydrochloric acid, longer hydrolysis leading to greater conversion to the diacid form. The monoacid derivative was purified from the diacids by silica gel column chromatography as previously described (Richter et al., 1990). BPD-MA was labeled with tritium by New England Nuclear (Boston, MA) according to the protocol previously published (Richter et al., 1990). Purity of the [3H]BPD products was determined by thin-layer chromatography (TLC) and biological activity measured by a standard cytotoxicity assay (Richter et a!., 1987). The specific activity of [3H]BPD-MA used was 5.465.9 mCii mg. [ 14C]BPD-MA was synthesized by Dr. David Dolphin, Department of Chemistry, University of British Columbia, by established methods (Richter et al., 1987). except that Protoporphyrin IX was reacted with [2,3-14C]dimethylacetylenedicarboxylate (sp. act. 44.0 mCi/mM), which 501

BETHA. ALLISONet al.

502

R ' = R 2 = C02Me

R 3 = (CH2)2C02Me

Figure 1. Structure of benzoporphyrin derivative. A and B designate the porphyrin rings at which fusion occurs to yield the ring A, ring B isomers. R3 represents the hydrolytic site for formation of the mono and diacid derivatives.

resulted in the incorporation of I4C into the cyclohexadiene ring of the BPD-MA product. The specific activity of the [14C]BPD-MA was 60.8 wCi/mg and purity was determined by TLC. All BPD analogs were stored in dimethylsulfoxide (DMSO) at -70°C at a concentration of 8 mg/me and were diluted immediately before use. The bioactivity of both [3H]BPD-MA and [I4C]BPD-MA was tested by in vitro cytotoxicity testing on cell lines (Richter et al., 1988). Both compounds had similar activity. Distribution of BPD in plasma. Two milliliter samples of human plasma were incubated for 18 h at 4°C in the presence of 100 pg of [,H]BPD-MA. The samples were then applied to a BIO-GEL A 5.0 M chromatographic column (90 X 1.5 cm) and eluted with 0.15 M NaCI, 10 mM Tris-HC1, 0.01% EDTA, 0.05% NaN,, pH 7.4, at 10 mUh. Eluate from the column was collected in 2.5 me fractions. Each column fraction was assayed for protein content by measuring absorbance at 280 nm. [3H]BPDMA content was assessed by diluting 100 of each fraction in 5 me Aquasol (New England Nuclear, Boston, MA) before counting in a Packard Tri-Carb 4550 liquid scintillation counter. Calibration of the column with human ['251]VLDL, -LDL, and -HDL allowed for identification of the resulting peaks (Pritchard et al., 1988). In other experiments, density gradient ultracentrifugation was used to study the association of [I4C]BPD-MA and -DA with plasma lipoproteins (Kelley and Kruski, 1986). Initially BPD was mixed with human plasma and incubated for 18 h at 4°C. This plasma was then adjusted to a density of 1.21 g/me by the addition of solid KBr. A step gradient was prepared using stock KBr density solutions at 1.006, 1.019, and 1.063 g/me. The density solutions were layered into the bottom of centrifuge tubes manually using a glass syringe and a narrow bore needle. The least dense solution was added first, so that denser solutions progressively pushed the lighter solutions to the top of the tube. The 1.21 glme adjusted plasma containing BPD was then layered into the bottom of the tube. Separation of the lipoproteins was accomplished by centrifugation in a Beckman SW 41 rotor for 24 h at 40 000 rpm and 15°C. By puncturing the tube below the most dense lipoprotein band and pumping the solution out, 0.5 me fractions were collected from the gradients. The protein content of each fraction was monitored by measuring absorbance at 280 nm. One-hundred microliters of each fraction was mixed with 5 me Aquasol (New England Nuclear) and counted in a liquid scintillation counter. The contribution of albumin binding to the BPD recovered in the HDL fraction was determined by a modification of the above sequential flotation protocol. After incubating the [3H]BPD-MA with plasma for 18 h at 4°C the density of this mixture was then adjusted to 1.21 g/me by the addition of solid KBr. Centrifugation at 40 000 rpm for 48 h resulted in the separation of the lipoproteins from

other plasma proteins including albumin (Rude1 et al., 1974). The lipoprotein fraction, taken from the top of the tubes, and the lipoprotein depleted fraction were then counted to assess BPD-MA content. The density of the lipoprotein fraction was readjusted to 1.21 g/mt before addition to the step density gradient (1.006-1.0639 g/me) previously described. Using this method, BPD association with the individual lipoproteins could be determined in the absence of albumin. Preparation of plasma lipoproteins. Lipoproteins were isolated from fresh human plasma by preparative ultracentrifugation. Three fractions were recovered by sequential flotation (Have1 et al., 1955), namely very low density lipoprotein (VLDL, density < 1.006 glme), low density lipoprotein (LDL, density 1.019-1.055 plrne), and high density lipoprotein (HDL, density 1.063-1.21 @me). The purity of each fraction was determined by agarose gel electrophoresis (Nobel, 1968). The total lipoprotein concentration was estimated by analysis of protein content (Lowry et al., 1961). Animals and dose of BPD. Biodistribution studies were performed in mature DBA/2J mice bearing the M1 ( D B N 2 methylcholanthrene induced rhabdomyosarcoma) tumor (Richter et al., 1990). Each mouse received 80 pg [3H]BPD-MA in either 0.1 me Tris:EDTA buffer (0.15 M NaCI, 10 mM Tris:HCI, 0.01% EDTA, 0.05% NaN,, pH 7.4) containing 10% DMSO or the appropriate lipoprotein solution containing 10% DMSO. This dose of BPD-MA had been previously shown to be effective in PDT of M1 tumors in DBN2J mice (Richter et al., 1988). Cures with BPD-MA can be achieved in this tumor model at doses as low as 2.5 mg/kg and light doses of 180 J/cm2. When using [14C]BPD-MA, each mouse received 100 pg BPD in similar solutions. Biodistribution of [l4C]BPDMA was analyzed to verify the results obtained with PHIBPD-MA. Because the specific activity of the I4C label was relatively low, a higher dose was injected than the dose of YHIBPD-MA. Previous experiments with [,H]BPD-MA had shown that doses ranging from 20 to 100 pg per mouse gave essentially identical biodistribution ratios. Studies done using 80 pg of 14C-labeled BPD gave biodistributions very similar to the mice reported here which received 100 pg. However, the former group gave such low counts at 24 h that the dose level was increased to provide more reliable counts. HDL and VLDL were used at 1 and 0.1 mg/me, respect-. ively, for biodistribution studies with both [I4C]BPD-MA and [,H]BPD-MA. LDL was used at 2 mg/me in experiments with both isotopes. [,H]BPD-MA or [14C]BPDMA was mixed with each purified lipoprotein fraction and incubated for 30 min at 37°C before intravenous injection into the tail vein. Each mouse received a dose of 4-5 mgl kg body weight. Immediately after injection, the mice were kept in the dark until death. They were allowed to eat and drink ad libitum. Biodistribution of BPD. Tumor bearing mice were injected with one of the lipoprotein-BPD mixtures described above. At 3, 8 or 24 h post-injection, mice were killed by cervical dislocation under light ether anesthesia and samples of blood, brain, heart, intestine, kidney, liver, muscle, skin, spleen, lymph node, feces, urine, bone marrow and tumor tissue were excised. Samples were placed in 7 me vials, minced and the wet weight or volume was determined. In addition, the total wet weight of each tumor was determined before duplicate samples were prepared for counting. Processing of samples was as previously described by Richter et al. (1990). Briefly, samples were solubilized in 1 me Protosol (New England Nuclear) for 3 days at 50°C. The solubilized samples were bleached with 100 p e of 30% hydrogen peroxide and mixed with 5 me of Econofluor (New England Nuclear). After 3-4 h adaptation in the dark, samples were counted in a Packard Tri-Carb 4550 liquid scintillation counter. DPM were subsequently

'

503

The plasma- and biodistribution of benzoporphyrin derivative converted to nanograms (ng) of [,H]BPD-MA or [I4C]BPD-MA per mg tissue. Values are expressed as a percentage of total BPD administered. Values reported represent the mean value of samples from 3 to 9 mice. Significant differences between the mean values of the different treatments were established by the Student's ttest.

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The elution profile of [3H]BPD-MA in plasma from the Biogel A5.OM column is presented in Fig. 2. This chromatographic procedure results in resolution of the three main lipoprotein classes. The profile of [3H]BPD-MA indicated that the majority eluted with H D L and albumin, some with LDL, and a small amount with the other plasma proteins which eluted after the H D L peak. VLDL bound very little or no [3H]BPD-MA. Poor resolution of H D L from albumin suggested that albumin binding might contribute to the apparent H D L binding. Therefore, we performed a density gradient separation of plasma which had been premixed with BPD-MA. Better resolution of albumin and H D L was accomplished with this technique (Fig. 3). The large protein peak in the first fractions included albumin, however, only 6% of the total [3H]BPDM A added was recovered in these fractions. BPDM A associated mainly with the HDL fraction (%YO), while recovery with the L D L and VLDL fractions appeared to be progressively less (15 and 3%, respectively). In a separate experiment, albumin was removed from the plasma lipoprotein fraction by ultracentrifugation of plasma at 1.21 glmC. When BPD-MA was added before this separation, 82% of total [14C]BPD-MA added was recovered in the plasma lipoprotein fraction and 5% in the lipoprotein deficient fraction. When albumin and other serum proteins were separated from the lipoprotein frac-

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Figure 3. Density gradient of [,H]BPD-MA and human plasma. 100 pg of ['HIBPD-MA was added to 3 mE of human plasma and incubated at 4°C for 18 h. This plasma sample was adjusted to a density of 1.21 glml by the addition of solid KBr and added to a KBr density gradient (1.0061.063 g/mC). The gradient was centrifuged at 40 OOO rpm for 24 h. 0.5 me fractions were collection from the gradient and assayed for protein and PHIBPD-MA content. tion before the addition of BPD, a shift in binding was observed. The majority of BPD-MA was still recovered with H D L (38% of total [14C]BPD-MA added); however, in this case, the association with L D L and VLDL fractions appeared to be equivalent (17% with L D L and 18% with VLDL, Fig. 4). Similar studies with BPD-DA added to the lipoprotein fraction indicated that a higher percentage associated with H D L (54%) and that VLDL bound slightly more than LDL (20 and 13%, respectively, Fig. 5). Polyacrylamide gel electrophoresis of the H D L fractions from these density gradients confirmed that very little albumin was present (data not shown). Thus, the association of BPD-MA and BPD-DA with H D L was not due to albumin contamination. Similar results were obtained in these experiments with both [3H]BPD-MA and [I4C]BPD-MA.

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Figure?.. Elution profile of Biogel A5.OM column. 100 pg of [3H]BPD-MAwas added to 2 me human plasma, incubated at 4°C for 18 h and then loaded onto the column. This sample was eluted at 10 me/h with 0.15 M NaCI, 10 m M Tris-HC1, 0.01% EDTA, 0.05% NaN,, pH 7.4. 2.5 me fractions were collected and assayed for protein content and [,H]BPD-MA content.

Figure 4. Density gradient of [I4C]BPD-MAin the plasma lipoprotein fraction of human plasma. 100 pg of [I4C]BPD-MAwere added to 2 mC of the 1.21 g/mC fraction of plasma, incubated for 18 h at 4°C and added to a KBr density gradient (1.0061.063 g/mC). The gradient was centrifuged at 40 000 rpm for 24 h. 0.5 mC fractions were collected from the gradient and assayed for protein and [I4C]BPD-MA content.

BETH A . ALLISON et al.

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Figure 5 . Density gradient of [I4C]BPD-DAin the plasma lipoprotein fraction of human plasma. 100 pg of [I4C]BPD-DA were added to 2 me of the 1.21 g/mt fraction of plasma, incubated for 18 h at 4°C and added to a KBr density gradient (1.006-1.063 g/me). The gradient was centrifuged at 40 000 rpm for 24 h. 0.5 me fractions were collected from the gradient and assayed for protein and [14C]BPD-DA content.

Biodistribution of BPD-MA The effect of precomplexing BPD-MA with plasma lipoproteins on the subsequent accumulation of radioactivity in tumor tissue was measured after 3, 8 and 24 h (Fig. 6). At 3 h, precomplexing BPDMA with LDL led to a significantly (P < 0.05) greater tumor deposition than BPD administered in aqueous solution. By 8 h, the amount of BPD-MA in the tumor was decreased in most treatment cases; however, the HDL mixture still resulted in enhanced deposition ( P < 0.05). By 24 h, clearance from the tumor had taken place with all three lipoprotein mixtures, as well as BPD in aqueous solution. As expected,the serum control led to accumulation in the tumor, which was roughly an average of the three isolated lipoproteins. The ratios of the mean percentage of administered BPD deposited in the tumor to that deposited

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in skin are shown in Fig. 7. At 3 h, the tumor : skin ratio for BPD in aqueous solution was consistently between 2 and 3. At this time point, precomplexing with both LDL and HDL led to significantly higher tumor to skin ratios (5.1 and 4.1, respectively). By 8 h, this ratio was still increasing with HDL (4.9), but the effect was no longer observed with LDL. After 24 h, the lipoprotein mixtures showed no advantage over BPD-MA in aqueous solution, with respect to tumor to skin ratio. The mean percentages of total [14C]BPD-MA administered, which was found to accumulate in various tissues, are presented in Tables 1 and 2. At all time points, accumulation was highest in the liver, kidney and spleen and lowest in bone marrow and brain. Association of BPD-MA with any of the three lipoproteins led to a higher blood level at 3 and 8 h than BPD alone (Tables 1 and 2). By 8 h this higher circulating level of BPD-MA was reflected in slightly higher deposition in most tissues in the presence of the lipoproteins. In all treatment cases, tissue associated radioactivity declined slowly with time. When BPD was delivered in aqueous solution, eliminaticn in the urine was approx. 10 times greater at 3 h than that at 8 h. Conversely, in the presence of lipoproteins, elimination of BPD in the urine at 3 h was lower than that at 8 h. Clearance in the feces was higher when BPD was delivered precomplexed with lipoproteins rather than in aqueous solution.

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Figure 6. Accumulation of [3H]BPD-MAin tumor tissue. The percent of total BPD administeredwhich accumulated in tumor tissue is presented. Each point represents the average value observed in 3-9 mice. Tumor tissue samples were excised at 3, 8 and 24 h post-injection of [3H]BPDMA.

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Figure 7 . Tumor : skin ratios with ["HIBPD-MA. Average (3-9 mice) accumulation of [3H]BPD-MA in tumor tissue was divided by the average accumulation in skin. Tumor and skin samples were excised at 3, 8 and 24 h post-injection of ['HIBPD-MA.

The plasma- and biodistribution of benzoporphyrin derivative

505

Table 1. [I4C]BPD-MA biodistribution results at 3 h Tissue Blood Brain Heart Intestine Kidney Liver Muscle Spleen Lymph node Feces Urine Gall bladder Bone marrow Tumor Skin

BPD

BPD & LDL

BPD & HDL

BPD & VLDL

2.34 f 0.12 0.66 f 0.02 2.34 f 0.01 3.35 f 2.87 3.92 f 0.17 25.27 t 1.57 2.50 t 2.16 10.37 f 3.46 1.95 f 1.52 2.39* 551.84 t 310.40 12.25 '. 9.15 0.01 f 0.00 2.70 2.12 1.61 f 1.08

4.42 f 0.43 0.53 f 0.06 3.67 t 0.24 7.64 t 2.69 11.22 f 7.35 21.90 f 4.89 3.89 2 3.79 21.46 f 3.95 18.58 f 20.52 42.27 t 32.14 147.92 t 58.56 303.84 t 446.58 0.12 f 0.07 13.36 f 6.55 1.10 t 0.22

2.69 t 0.38 0.26 t 0.04 2.66 2 0.63 2.49 t 0.56 3.05 f 2.29 11.58 t 8.08 0.70 f 0.18 6.16 t 1.27 4.26 t 4.46 17.43 f 8.80 109.68 f 94.80 26.06 f 10.00 0.01 2 0.00 2.38 t 1.14 0.78 f 0.42

4.35 f 1.40 1.66 f 1.87 8.02 f 8.93 2.41 f 0.12 16.12 f 19.48 25.73 f 21.15 1.06 t 0.31 15.33 f 4.53 4.94 f 2.21 59.18 t 69.6 267.04 f 184.08 26.89 t 4.82 0.19 f 0.19 5.66 f 2.54 2.02 f 0.99

*

Values represent BPD accumulated in tissue expressed as mean percent of total BPD administered x *One sample only. Table 2. [14C]BPD-MA biodistribution results at 8 h Tissue Blood Brain Heart Intestine Kidney Liver Muscle Spleen Lymph node Feces Urine Gall bladder Bone marrow Tumor Skin

BPD

BPD & LDL

BPD & HDL

1.26 2 0.07 0.55 f 0.23 1.97 t 0.82 0.96 t 0.18 2.31 f 0.19 17.49 t 1.52 0.55 f 0.17 8.90 t 4.16 0.77 t 0.35 50.67 t 69.29 50.00 f 2.55 2.43 f 0.52 0.28 f 0.43 1.49 f 0.29 0.47 f 0.17

5.09 f 5.90 0.43 t 0.23 1.04 -+ 0.17 1.18 f 0.53 2.40 f 0.75 9.39 t 1.42 0.78 t 0.31 6.42 f 1.14 3.91 f 2.04 122.24 f 145.04 719.52 f 370.72 22.21 f 13.02 0.15 f 0.21 3.50 f 1.02 1.23 t 0.12

2.33 f 0.07 1.15 t 0.82 1.81 t 0.46 1.37 f 0.17 6.49 f 6.07 11.97 f 0.52 0.45 f 0.03 5.23 f 1.32 2.54 f 0.94 274.48 f 117.76 720.40 t 157.28 7.33 f 6.14 0.00 f 0.00 2.54 t 0.53 1.21 t 0.69

BPD & VLDL

2.42 f 0.76 0.30 t 0.08 1.30 f 0.39 1.53 f 0.41 3.07 f 0.70 24.50 f 6.22 0.42 f 0.08 10.22 f 5.40 1.68 f 0.22 297.36 f 214.80 111.12 f 55.92 11.63 f 8.19 0.01 f 0.00 2.77 t 0.52 1.37 f 0.58

~~

Values represent BPD accumulated

in

tissue as mean percent of total BPD administered

has been proposed for several other porphyrins (Reyftmann et al., 1984; Jori et al., 1984; Kessel, 1986). Chromatographic separation of plasma lipoprotein associated BPD-MA suggested that the majority of BPD binds to HDL with significantly less binding to LDL and VLDL. These results are very similar to those previously observed by Kessel (1986) and Barel et al. (1986)--usingdifferent porphyrins. Because most methods of lipoprotein separation do not produce HDL which is free of albumin, we decided to further investigate the contribution of albumin to the recovery of BPD and HDL. Resolution of albumin and HDL by density gradient ultracentrifugation demonstrated that little BPD was binding to albumin. When albumin and other serum proteins were removed from the plasma lipoproteins by ultracentrifugation, over 80% of the BPD-MA

in the plasma sample was recovered with the plasma lipoproteins. When albumin was removed from the plasma sample before the addition of BPD, there was increased recovery in the VLDL fraction. These results suggest that albumin might compete with VLDL for the binding of BPD. The association of BPD with HDL and LDL, however, was comparable in the absence or presence of albumin. Polyacrylamide gel electrophoresis of the HDL containing fractions showed that albumin contamination in these fractions was negligible and therefore the association with HDL was concluded to be genuine. The slight change in hydrophobicity contributed by the additional carboxylic acid group in the diacid form of BPD led to increased association with HDL and VLDL. LDL association was slightly decreased. In another study (Richter et al., unpublished data),

506

et al. BETHA . ALLISON

we have shown that BPD diacids are cleared from the body at a faster rate than the monoacids. It is possible that these differences in lipoprotein association contribute to this property. Kessel (1989) performed density gradient plasma distribution studies with BPD using a different method (Chung et al., 1986). In agreement with our results, HDL was observed to bind the majority of both BPD-MA and BPD-DA. Similarly, a decrease in LDL binding was reported in the case of BPDDA. Because albumin and other serum proteins were present in Kessel’s density gradients, the reported VLDL binding was low. These results agree with our findings that little BPD associates with VLDL in the presence of albumin. Only upon the removal of albumin and other serum proteins was a significant portion of the BPD recovered with the VLDL fraction. The accumulation of BPD-MA in tumors was enhanced by LDL association at 3 h post-injection, as previously described for Hp by Barel et al. (1986). HDL association also had an affect on BPDMA deposition at this time point, but it was not as marked. By 8 h post-injection, clearance of BPDMA from the tumor had begun, with differing rates depending upon the plasma fraction mixture of BPD-MA. There was one exception-that the association of BPD-MA with HDL led to increased accumulation in the tumor at least up to 8 h postinjection. This finding may be a function of the longer half-life of HDL in circulation as compared to VLDL and LDL. Further experiments are required to investigate this effect. By 24 h the BPD which had accumulated in tumors had decreased regardless of the mode of delivery. The accumulation of photosensitizer in the skin is of great interest-the main side effect of PDT with HPD and DHE is phototoxic reactions in areas of skin exposed to daylight. Experiments by Richter et al. (1988) indicate that BPD-MA does not cause severe photosensitivity of the skin after 24 h. However, [3H]BPD-MA in aqueous solution distributes between the tumor and skin with a ratio greater than 2 at 3 h post-injection. The association of BPDMA with lipoproteins increases this ratio. In particular, LDL and HDL lead to ratios as high as 5 after 3 h. Delivery of BPD in association with these two lipoproteins should provide for increased deposition in the tumor and less potential for skin sensitivity at this time point. However, clearance of BPDMA from the skin was slower when the drug was administered after precomplexing with lipoproteins. This reduced the advantage over aqueous BPD after 8 and 24 h. The association of BPD-MA with any of the three lipoproteins led to increased circulating levels of the drug. This may be due to the differences in the clearance of BPD observed. As would be expected, lipoprotein association decreased excretion in the urine at 3 h post-injection. However, this prolonged

circulation of lipoprotein associated BPD resulted in higher levels in the urine and feces after 8 h. These high levels of radioactive drug observed in feces probably reflect the metabolism of the lipoproteins via the liver. Lipoprotein associated BPD may be secreted in bile acids into the digestive tract to be eliminated in the feces. These studies indicate that delivery of BPD-MA to tissues follows the principles proposed for other porphyrins such as HP and PhotofrinB. Both the monoacid and diacid forms of BPD associate with lipoproteins when mixed with plasma. Association with lipoproteins, in particular LDL, enhanced accumulation of this poprhyrin at the tumor site. Lipoprotein association also affected clearance of the drug. At 8 h post-injection, HDL associated BPD remained in tumors while clearance had begun with all other treatments. Coupled with the high cytotoxicity of BPD (Richter et al., 1987), lipoprotein association would be expected to lead to an enhanced therapeutic effect upon irradiation of tumors. PDT of tumor bearing mice will be performed to investigate this possibility. Further studies into the mechanism of lipoprotein enhanced delivery will also be performed. These studies may give insight into the site and mechanism of porphyrin uptake and action. Manipulation of these mechanisms could provide for selective tumor delivery and enhanced tumor necrosis following PDT.

Acknowledgements-This work was supported by grants from the B.C. Heart Foundation and a University/Industry Grant from the Natural Science and Engineering Research Council of Canada. Beth A. Allison was supported by a scholarship from the Science Council of British Columbia. Excellent technical assistance was provided by Lisa Macht and Richard Sevigny.

REFERENCES

Barel, A,, G. Jori, A. Perin, P. Romandini, A. Pagnan and S. Biffanti (1986) Role of high-, low- and very-lowdensity lipoproteins in the transport and tumor-delivery of hematoporphyrin in vivo. Cancer Lett. 32, 145-150. Chung, B., J. Segrest, M. Ray, J. Brunsell, J. Hokanson, R. Krauss, K. Beaudrie and J. Cone (1986) Single vertical spin density gradient ultracentrifugation. Mefh. Enzymol. 128, 181-209. Havel, R. J., H. A. Eder and J. H. Bragdon (1955) Distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J . Clin. Invest. 34, 1345-1353. Jori, G., M. Beltramini, E. Reddi, B. Salvato, A. Pagnan, L. Ziron, L. Tomio and T. Tsanav (1984) Evidence for a major role of plasma lipoproteins as hematoporphyrin carriers in vivo. Cancer Lett. 24, 291-297. Kelley, J. K. and A. W. Kruski (1986) Density gradient ultracentrifugation of serum lipoproteins in a swinging bucket rotor. Merh. Enzymol. 128, 170-181. Kessel, D. (1986) Porphyrin-lipoprotein association as a factor in porphyrin localization. Cancer Lett. 33, 183-188.

The plasma- and biodistribution of benzoporphyrin derivative Kessel, D. (1989) In vitro photosensitization with a benzoporphyrin derivative. Photochem. Photobiol. 49, 579-582. Lowry, 0. H., M. J. Rosebrough, A. L. Farr and R. J. Randall (1961) Protein measurement with the folinphenol reagent. J. Biol. Chem. 193, 265-275. Nobel, R. P. (1968) Electrophoretic separation of plasma lipoproteins in agarose gel. J. Lipid Res. 9, 693-700. Pritchard, P. H., R . McLeod, J. Frohlich, M. C. Pork, B. J. Kudchodkar and A. G. Lacko (1988) Lecithin:cholesterol acyltransferase in familial HDL deficiency (Tangier disease). Biochim. Biophys. Acta 958, 227-234. Reyftmann, J. P., P. Morliere, S. Goldstein, R . Santus, L. Dubertret and D. Lagrange (1984) Interaction of human serum low density lipoproteins with porphyrins: a spectroscopic and photochemical study. Photochem. Photobiol. 40, 721-729. Richter, A. M., S. Cerruti-Sola, E. D. Sternberg, D. Dolphin and J . G. Levy (1990) Biodistribution of triti-

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ated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer, in normal and tumor bearing mice. J . Photochem. Photobiol. 5, 231-244. Richter, A. M., B. Kelly, J. Chow, D. J . Liu, G. H. N. Towers, D. Dolphin and J. G. Levy (1987) Preliminary studies on a more effective phototoxic agent than hematoporphyrin. J. Natl. Cancer Inst. 79, 1327-1332. Richter, A. M., E. Sternberg, E. Waterfield, D. Dolphin and J. G. Levy (1988) Characterization of benzoporphyrin derivative, a new photosensitizer. Proc. SPIE-Int. SOC. Op. Engng 997, 132-138. Rudel, L. L., J. A. Lee, M. D. Morris and J. M. Felts (1974) Characterization of plasma lipoproteins separated and purified by agarose-column chromatography. Biochem. J. 139, 89-95. Zhou, C., C. Milanesi and G. Jori (1988) An ultrastructural comparative evaluation of tumors photosensitized by porphyrins administered in aqueous solution, bound to liposomes or to lipoproteins. Photochem. Photobiol. 48, 487-492.