Journal of Photochemistry and Photobiology, B, Biology, 5 (1990) 231 - 244

231

BIODISTRIBUTION OF TRITIATED BENZOPORPHYRIN DERIVATIVE (3H-BPD-MA), A NEW POTENT PHOTOSENSITIZER, IN NORMAL AND TUMOR-BEARING MICE ANNA M. RICHTER

Quadra Logic Technologies, 520 West 6th Avenue, Vancouver, British Columbia V5Z 4H5 (Canada) SUSANNA CERRUTI-SOLA

Department of Animal Pathology, Veterinary Institute, Torino (Italy) ETHAN D. STERNBERG, DAVID DOLPHIN and JULIA G. LEVY ~

Departments of Microbiology and Chemistry, University of British Columbia, 61 74University Blvd., Vancouver, British Columbia V6T 1 W5 (Canada) (Received April 27, 1989; accepted July 10, 1989)

Keywords. Photosensitizer, photodynamic therapy, cancer therapy, benzoporphyrin derivative, biodistribution, tumor localization, tissue clearance. Summary The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (mastocytoma) or M1 (rhabdomyosarcoma) tumor-bearing DBA/2J mice. A dose of 80/~g of 3H-BPD-MA was determined at 3, 24, 48, 72, 96 and 168 h post injection. The following tissues were tested: blood, brain, heart, intestine, kidney, lung, liver, muscle, skin, stomach, spleen, thymus and tumor. The biodistribution of 3H-BPD-MA in normal and tumor-bearing mice was comparable overall. 3H-BPD-MA localized in tumors better than in other tissues except kidney, liver and spleen. The tumor to tissue ratios were in the range 1.5 - 3 at 24 h post injection and increased further during the next 72 h. The highest levels of 3H-BPD-MA were observed in all tissues at 3 h post injection and decreased rapidly during the first 24 h. After 24 h the clearance from tissues was rather slow. The preliminary clearance data obtained in a group of five normal mice indicated that the majority of the injected dose {60%) cleared from the body via the bile and feces, while only about 4% cleared via kidneys and urine. Studies in which 3H-BPD-MA was extracted from tumor, kidney and liver 3 and 24 h after injection showed that, at 3 h, all the photosensitizing activity in tumor was retained. At 24 h only 39% of the activity was retained and considerably less active material was present in liver and kidney. tAuthor to w h o m

correspondence should be addressed. Elsevier Sequoia/Printed in The Netherlands

232

1. Introduction Early observations of the light-mediated cytotoxicity of porphyrins [1] and of the accumulation of porphyrins in malignant tissues accompanied by the emission of red fluorescence [2, 3] led to the development of photodynamic therapy (PDT) for cancer. The natural tendency of hematoporphyrin to accumulate in tumors was inproved further by its modification with a mixture of sulfuric and acetic acids followed by neutralization with alkali [4]. The resulting mixture of porphyrins has been named hematoporphyrin derivative (HPD). Although, at first, HPD has been considered for use in t u m o r detection due to its red fluorescence, its therapeutic potential was soon realized. Shortly after Dougherty e t al. [5] reported the complete eradication of a transplanted mammary t u m o r in mice using HPD and red light, HPD was tested in clinical situations. At this time, a large number of patients have been tested with HPD, resulting in many encouraging reports (for a state-of-the-art report see refs. 6 and 7). The main tumor-localizing components of HPD have been identified as hematoporphyrin dimers and oligomers joined by labile linkages of ether or ester type [8 - 10]. The somewhat purified fraction o f HPD, containing the tumor-localizing components, has been c o m m o n l y referred to as dihematoporphyrin ether (DHE) and its use has allowed the clinically active dose of HPD to be reduced by almost one-half. A photosensitizer is most efficiently activated by light at the wavelength at which it absorbs most effectively. Theoretically, the photosensitizer most suitable for PDT, in addition to its tumor-localizing ability, should absorb light strongly at 700 - 850 nm or 1000 - 1100 nm; these are the wavelengths which best penetrate tissues. Although DHE has been used extensively in the clinical setting, there is considerable interest and basic research is being carried o u t to examine new photosensitizers with properties distinct from those of DHE. We have recently reported [11] the results of in vitro cytotoxicity tests with another photosensitizer from the porphyrin group named benzoporphyrin derivative (BPD). This c o m p o u n d exhibits an absorption peak at 692 nm and under the same experimental conditions is 10 - 70 times more phototoxic than hematoporphyrin towards various cell lines. Since then the

n3c. R

I

CH3 ~

R5 HsC

R1

R2 : C02Me R3 : (CH2)2CO2Me or

(CH2)2002H Fig. i. Structure of benzoporphyrin derivative-monoacid, ring A (BPD-MA).

233 c o m p o u n d has been further modified resulting in an even more cytotoxic c o m p o u n d named benzoporphyrin derivative-monoacid, ring A (BPD-MA, Fig. 1). This c o m p o u n d has a tumor-localizing ability such that there are higher levels in the t u m o r than in some normal tissues such as skin and muscle. We have studied the biodistribution of tritiated BPD-MA in normal and tumor-bearing mice and the results are reported here. Some preliminary data concerning plasma clearance and elimination of 3H-BPD-MA from the body are also reported.

2. Materials and methods 2.1. Photosensitizer The synthesis of BPD as described earlier [11] resulted in a diacid derivative of the chromophore with a ring fused on ring B of the porphyrin. The BPD isomer with a ring fused on ring A of the porphyrin was modified to a monoacid derivative by decreasing the length of hydrolysis with 25% hydrochloric acid from 5 h to 20 min. The monoacid derivative (Fig. 1) was then purified by passage over a silica gel chromatography column and elution with dichloromethane-ethylacetate-acetic acid (55:44:1). The second and major fraction off the column was collected. After evaporation of the solvent the product was dissolved in dichloromethane, precipitated with hexane and dried in vacuum. The product thus obtained was named benzoporphyrin derivative-monoacid, ring A (BPD-MA). Since the chromophore was n o t changed, BPD-MA exhibited, like BPD, an absorption peak at 692 nm. The extinction coefficient for BPD-MA in dichloromethane is 37 000 M-1 cm -1 at 688 nm. BPD-MA was labeled with tritium by New England Nuclear (NEN, Boston, MA, U.S.A.) according to the following prot6col. Approximately 100 mg of the c o m p o u n d was dissolved in 2 ml of dimethylformamide (DMF). Tritiated water (15 ml) and 5% Rh-A1203 (40 mg) were added to this. The solution was heated to between 80 and 100 °C overnight after which it was filtered using methanol to remove all residual compounds from the catalyst. After evaporation the product was redissolved in a small a m o u n t of methanol to exchange out any residual labile tritium. The labeled product, after evaporation of methanol, was dissolved in dimethylsulfoxide (DMSO). The final solution was frozen for storage and shipment. The labeling efficiency was 1% - 2% at the methine and methane hydrogens across the molecule. As this level of labeling is n o t easily detected by mass spectrometry, we repeated the procedure using deuterated water under more severe conditions ( 1 2 0 - 130 °C for 2 days) and saw a level of deuterium incorporation of about 20% with some concurrent hydrolysis to the corresponding diacid BPD. We checked the stability of the label by subjecting the NEN tritiated material to column chromatography on silica gel using elution with dichloromethane-ethylacetate-acetic acid and found no significant loss of

234 label. Similarly, an analysis by thin-layer chromatography (TLC) showed that the label was associated with the BPD-MA band. We also checked for photosensitizing activity of tritiated BPD-MA using a standard cytotoxicity assay on P815 cells as described earlier [11]; it was found t h a t it was fully active (LDs0 as determined in P815 cells was 5.7 ng m1-1 and 6.0 ng m1-1 for 3H-BPD-MA and cold BPD-MA respectively). All biodistribution studies reported here were performed within 4 weeks of obtaining the tritiated material; the tritium was associated with the BPD throughout this period. The specific activity was 5.9 mCi mg -1 SH-BPD-MA and the concentration was 7.625 mg 3H-BPD-MA m1-1 DMSO. The labeled c o m p o u n d was stored at 4 °C. Appropriate amounts of 3H-BPD-MA were diluted with phosphate-buffered saline (PBS) to a concentration of 800 pg m1-1 immediately before injection into animals. The c o m p o u n d v~as protected from light at all times. All solvents, reagent grade, were obtained from Fisher Scientific {Vancouver, Canada).

2.2. Animals and dose o f 3H-BPD-MA All tests were performed on mature DBA/2J female mice weighing approximately 20 g. Each mouse received 80 pg 3H-BPD-MA in 0.1 ml PBS containing 10% DMSO intravenously (i.v.) via the tail vein. Thus each mouse received a dose of 4 mg kg-1 body weight. I m m e d i a t e l y after injection the animals were kept in the dark for 3 h and then transferred to the animal facility with alternate periods of 12 h light and 12 h dark. They were allowed to eat and drink ad libitum.

2.3. Tumors The cell lines, P815 (DBA/2 mastocytoma) and M1 (DBA/2 methylcholanthrene-induced rhabdomyosarcoma) have been maintained in our laboratory for 10 years. P815 cells were recovered from ascites, washed with PBS and injected subcutaneously in the right flank at a concentration of 104 cells per mouse. After 15 days the tumors were 8 - 12 mm in diameter and the mice were used in 3H-BPD-MA biodistribution studies. M1 cells were recovered from solid tumors by pressing excised tissue through a wire mesh. They were washed in PBS and injected subcutaneously in the right flank at a concentration of 104 cells per mouse. After 16 days the tumors ranged in size from 5 to 10 mm in diameter and the mice were used in 3H-BPD-MA biodistribution studies.

2.4. 3H-BPD-MA b iodistribution studies Three groups of mice were used in this study. Group I consisted of 18 normal healthy mice, group II consisted of 18 P815 tumor-bearing mice and group III consisted of nine M1 tumor-bearing mice. Each mouse received an

235 intravenous injection of 80 pg 3H-BPD-MA. At 3, 24, 48, 72, 96 and 168 h post injection (groups I and II) and at 24, 96 and 168 h post injection (group III), three mice per group were sacrificed b y cervical dislocation under light ether anesthesia and blood and tissue samples were collected for the determination of 3H-BPD-MA content.

2.5. Plasma clearance and elimination o f 3H-BPD-MA This experiment was designed to provide preliminary data concerning the fate of 3H-BPD-MA in the body. Five mice received 8 0 / J g of 3H-BPDMA intravenously, and samples of blood, urine and feces were collected before and at 1, 3, 5, 8, 24, 48, 72 and 96 h after the injection. In addition, at the same time intervals, all feces from the cage were collected and weighed in order to determine the total amount of feces produced during each period. At 96 h post injection the mice were sacrificed and samples of the tissues were tested for their 3H-BPD-MA content as described below. 2.6. The determination o f 3H-BPD-MA content in the samples The SH-BPD-MA content was determined in the following tissues listed in alphabetical order: blood, brain, gall bladder, heart, intestine, kidney, lung, liver, muscle, skin (ear), spleen, stomach, thymus and tumor. Samples of urine and feces were tested only from the mice in the plasma clearance and elimination study. Single samples were tested from all tissues except kidney, liver, spleen and tumor. These tissues were tested in duplicate. Blood and other contaminants were washed off the tissue samples with PBS. PBS was then dried off with blotting paper. The size of the liquid samples was determined by volume and ranged from 10 to 100 pl. The size of the solid samples was determined by wet weight and ranged between 15 and 100 mg (except for the gall bladder which usually weighed much less). The total wet weight of each t u m o r was determined and then duplicate or multiple samples were prepared for testing. All samples, including blood, urine and feces, were processed in an identical manner. The samples were placed in glass vials (7 ml) and after mincing the tissues, the samples were solubilized in 1 ml Protosol (NEN) for 3 days at 50 °C. The solubilized samples were bleached with 100 pl of 30% hydrogen peroxide and then mixed with 5 ml of Econofluor (NEN). After 3 - 4 h adaptation in the dark, the samples were counted in a Packard TriCarb 4550 liquid scintillation counter. The number of counts per minute (c.p.m.) was converted to the number of disintegrations per minute (d.p.m.) using quench curves constructed separately for each tissue. Disintegrations per minute were subsequently converted to micrograms of SH-BPD--MA and expressed per gram of w e t tissue, or per milliliter of either blood or urine. The efficiency of the m e t h o d was tested by adding a known amount of 3H-BPD-MA to tissue samples. These samples were then processed according to the routine protocol, and the a m o u n t of radioactivity was determined using the appropriate quench curves. The average error of the determination was 13%.

236

2.7. Extraction and testing o f 3H-BPD-MA from mouse tissues Some preliminary tests were carried out to correlate the radioactivity detected in tissues with the presence of active BPD-MA. The tissues with the highest content of 3H-BPD-MA, the liver, kidney and tumor, were chosen for testing. At 3 and 24 h post i.v. injection of 150 pg BPD-MA per mouse, containing 16% 3H-BPD-MA, the tissues were dissected and homogenized on ice in a small volume of ice-cold distilled water. The homogenates were freeze-dried and extracted with dichloromethane containing 5% methanol. The solvent was evaporated and the dry extracted material was dissolved in a small volume of DMSO and then diluted further with Dulbecco's minimum essential medium (DME). The whole procedure was carried out in dim light. Tissues of animals which did n o t receive any BPD-MA were extracted in the same manner and served as controls. Addition of 3H-BPD-MA to tissue homogenates followed by the extraction procedure resulted in about 50% recovery of radioactive material. Tissue extracts were checked for their radioactivity and for their photosensitizing activity. Samples of 50 pl were counted in a liquid scintillation counter as described above. The a m o u n t of radioactivity per sample, expressed in disintegrations per minute, was converted to the a m o u n t of BPD-MA using a conversion factor determined from the BPD-MA solution used for injection. Phototoxicity of extracts was determined by means of a cytotoxicity assay using P815 cells and the affinity protocol as described previously [11]. The a m o u n t of active BPD-MA in the extracts was calculated from the phototoxic activity of the extracts using a standard curve obtained with pure BPD-MA.

3. Results

3.1. Biodistribution o f 3H-BPD-MA The levels of SH-BPD-MA in the tissues of normal and tumor-bearing mice expressed in Micrograms per gram of wet tissue are presented in Table 1. Since the analysis of tissue extracts, as reported below, indicates that BPD-MA is metabolized in the tissues relatively quickly, we report the results obtained up to 48 h only. The a m o u n t of radioactivity determined in the gall bladder was related to the a m o u n t of bile present in the gall bladder. Thus the individual samples taken at the same time after the injection varied widely and the results are n o t tabulated. The data (not shown) suggest a very high accumulation of 3H-BPD-MA in the bile at 3 h post injection followed by a gradual decrease. In all tissues, including tumors, the highest concentration of the radioactive c o m p o u n d was present at the earliest time i.e. at 3 h post injection. At that time the lowest concentrations were observed in the brain and the highest in the liver, followed by spleen and kidney. The concentration of radioactive BPD-MA in blood at 3 h was higher in P815 tumor-bearing mice than in normal mice. The label disappeared from

237

TABLE 1 B i o d i s t r i b u t i o n a n a l y s i s o f 3 H - B P D - M A in n o r m a l ( g r o u p I) a n d P 8 1 5 ( g r o u p I I ) o r M1 ( g r o u p I I I ) t u m o r - b e a r i n g D B A / 2 J m i c e at v a r i o u s t i m e s a f t e r i n t r a v e n o u s i n j e c t i o n o f 80 p g 3 H - B P D - M A p e r m o u s e . T h e v a l u e s r e p r e s e n t t h e m e a n p l u s o r m i n u s t h e s t a n d a r d d e v i a t i o n ( S D ) o f t h e d e t e r m i n a t i o n s in t h r e e m i c e a n d are e x p r e s s e d in M i c r o g r a m s o f 3H-BPD-MA per gram of wet tissue

Tissue

Time after injection

Group I

Group H

Group III

(h) Blood a

3 24 48

3.44 + 0.32 0 . 9 1 + 0.07 0 . 5 0 -+ 0 . 0 8

5 . 6 1 -+ 0 . 1 4 1.03 + 0.22 0 . 7 4 +- 0 . 0 9

--

Brain

3 24 48

0 . 5 2 -+ 0 . 1 6 0.37 + 0 . 0 6 0 . 3 1 -+ 0 . 0 2

0 . 7 9 + 0.07 0.49 + 0.05 0 . 3 5 + 0.07

-0 . 5 6 + 0.01 --

Heart

3 24 48

1.64 + 0.53 0 . 4 6 -+ 0.07 0.49 + 0.08

2.69 + 0.19 0.87 + 0 . 0 9 0.61 + 0 . 0 4

-0.86 + 0.14 --

Intestine

3 24 48

0 . 6 0 +- 0 . 1 1 0 . 4 5 + (~.14 0.37 -+ 0 . 0 3

1 . 2 0 -+ 0 . 1 1 0 . 8 0 -+ 0 . 0 6 0.53 + 0.04

-0.86 + 0.42 --

Kidney

3 24 48

3.94 + 0.84 2.47 + 0.33 2.06 + 0.26

6 . 1 0 + 1.11 3 . 1 8 +- 0 . 3 0 2.43 + 0.35

--

Liver

3 24 48

25.25 + 5.34 16.11 + 2.11 1 7 . 3 7 -+ 1.48

33.50 + 6.20 1 8 . 8 6 -+ 2 . 0 3 1 6 . 8 4 -+ 2.57

-1 5 . 0 3 -+ 1 . 5 2 --

Lung

3 24 48

1.87 + 0 . 1 4 0.59 + 0.16 0 . 6 5 -+ 0 . I 0

3.72 + 0.22 1 . 0 5 + 0.21 0 . 6 8 + 0.07

-1.07 + 0 . 2 4 --

Muscle

3 24 48

0 . 8 4 -+ 0 . 0 9 0.38 + 0.10 0 . 3 3 + 0.07

1.09 + 0.08 0.50 + 0.09 0.38 + 0 . 0 9

--

Skin

3 24 48

0 . 9 8 -+ 0 . 2 4 0.48 -+ 0 . 1 1 0.45 + 0.05

1.16 + 0.12 0 . 9 4 + 0.11 0 . 5 1 -+ 0 . 0 1

-0.54 + 0.01 --

Stomach

3 24 48

0 . 7 8 -+ 0 . 2 8 0.39 + 0.13 0.39 + 0.03

1 . 8 6 + 0.07 0.80 + 0.13 0 . 4 9 + 0.07

-0.74 + 0.14 --

Spleen

3 24 48

5.22 + 2.57 4 . 7 3 -+ 1 . 1 4 4.48 + 0.62

1 0 . 7 1 -+ 5 . 4 3 4.49 + 1.54 4.80 + 0.39

1.32 + 0.18 --

3.95 + 0.32 --

0.46 + 0.11 --

-2.3 --

+ 0.97

(continued)

238 TABLE 1 (continued) Tissue

Thymus

Time after injection (h) 3 24

48 Tumor

3 24 48

Group I

0.71 + 0.29 0.29 + 0.15 0.39 2 0.04

----

Group H

Group III

1.68 + 0.22 1.03 + 0.15 0.68 + 0.08

--

2.94 + 0.61 1.49 + 0.41 1.00 -+ 0.12

-1.25 + 0.16

1.07 + 0.37 --

--

aValues expressed in Micrograms of SH-BPD-MA per milliliter of blood. the b l o o d o f t u m o r - b e a r i n g m i c e at a slightly slower r a t e t h a n f r o m t h e b l o o d o f n o r m a l m i c e ; h o w e v e r , at 1 6 8 h, v e r y little label was p r e s e n t in t h e blood of either group. Overall t h e levels o f r a d i o a c t i v i t y w e r e higher in b o t h P 8 1 5 a n d M1 t u m o r s t h a n in t h e n o r m a l tissues w i t h t h e e x c e p t i o n o f t h e liver, spleen a n d k i d n e y . A t 3 h p o s t i n j e c t i o n o n l y , levels in t h e b l o o d a n d lung w e r e higher t h a n in t u m o r tissue. H o w e v e r , at 24 h these levels w e r e a l r e a d y b e l o w t h e levels in t h e t u m o r s . T h e c o n c e n t r a t i o n o f r a d i o a c t i v e m a t e r i a l in tissues was s h a r p l y r e d u c e d b e t w e e n 3 h a n d 2 4 h p o s t i n j e c t i o n w i t h t h e s h a r p e s t d r o p o b s e r v e d in t h e b l o o d , heart, lung a n d m u s c l e . This was f o l l o w e d b y a gradual decrease d u r i n g t h e n e x t 24 - 168 h. T h e clearance o f r a d i o a c t i v e m a t e r i a l f r o m s o m e tissues is s h o w n in Fig. 2. T h e rates o f clearance in o t h e r tissues w e r e interm e d i a t e b e t w e e n t h e rates f o r h e a r t a n d intestine. In t h e liver, t h e decrease a f t e r t h e first 24 h was v e r y slow a n d in t h e spleen s o m e a c c u m u l a t i o n o f t h e label b e t w e e n 96 a n d 168 h was o b s e r v e d . T h e r a d i o a c t i v i t y in t u m o r s d e c r e a s e d w i t h t i m e , b u t a t a s l o w e r r a t e t h a n in n o r m a l tissues. T h e ratios b e t w e e n the c o n c e n t r a t i o n s o f 3 H - B P D MA in t u m o r tissue a n d in n o r m a l tissues w e r e similar f o r P 8 1 5 a n d M1 t u m o r - b e a r i n g m i c e a n d increased w i t h t i m e a f t e r the injection. T h e t u m o r t o tissue ratios in P 8 1 5 t u m o r - b e a r i n g m i c e f o r s o m e tissues o f i n t e r e s t are p r e s e n t e d in T a b l e 2. 3.2. Plasma clearance a n d e l i m i n a t i o n o f a H - B P D - M A T h e m e a n s a n d s t a n d a r d d e v i a t i o n s o f t h e levels o f r a d i o a c t i v e m a t e r i a l ( e x p r e s s e d in disintegrations p e r m i n u t e p e r milligram or m i c r o l i t e r ) in b l o o d , urine a n d feces d e t e r m i n e d a t various t i m e intervals p o s t i n j e c t i o n are s h o w n in Fig. 3. T h e highest c o n c e n t r a t i o n s o f r a d i o a c t i v e m a t e r i a l w e r e o b s e r v e d in t h e earliest s a m p l e s o f b l o o d a n d urine. H o w e v e r , in t h e feces t h e r a d i o a c t i v i t y p e a k e d at 5 h ( t h r e e mice) or 8 h ( t w o m i c e ) p o s t injection. T h e r e d u c t i o n in t h e levels o f r a d i o a c t i v e m a t e r i a l in t h e b l o o d a n d urine s a m p l e s was m o s t

239 100"

J~ 03

>

--e- Blood

.J

50 ¸ &

"4-

Heart

-~

Intestine

-4-

Spleen

Liver

Oz. QQ

+ ¢o



i

24



i

48



,



i

72 96 Time (hours)

,

,

120

-

,

144

-

,

168

Fig. 2. The rate o f clearance o f 3H-BPD-MA f r o m tissues o f n o r m a l mice a f t e r i n t r a v e n o u s injection o f 80 /~g 3H-BPD-MA per m o u s e . The levels o f r a d i o a c t i v i t y ( d i s i n t e g r a t i o n s per m i n u t e per milligram o f w e t tissue) in tissues are e x p r e s s e d as t h e p e r c e n t a g e o f t h e level p r e s e n t in each tissue at 3 h p o s t injection. TABLE 2 The ratios b e t w e e n the levels o f r a d i o a c t i v i t y (disintegrations p e r m i n u t e per milligram o f w e t tissue) in t h e t u m o r and in n o r m a l tissues d e t e r m i n e d at various t i m e s p o s t i n j e c t i o n in P815 t u m o r - b e a r i n g mice (each value r e p r e s e n t s m e a n + S D as d e t e r m i n e d in three mice)

Tissue

Time post injection (h) 3

Blood Brain Heart Intestine Kidney Liver Lung Muscle Skin Spleen Stomach

0.52 3.76 1.09 2.42 0.47 0.09 0.79 2.68 2.57 0.31 1.57

24 + 0.10 + 1.11 + 0.17 + 0.28 + 0.02 + 0.01 + 0.13 + 0.39 -+ 0.73 + 0.07 + 0.15

1.45 3.06 1.71 1.85 0.47 0.08 1.55 2.98 1.64 0.34 1.89

48 -+ 0.23 -+ 0.75 + 0.41 + 0.48 + 0.16 + 0.02 + 0.87 +- 0.72 + 0.65 + 0.05 + 0.55

1.37 2.92 1.63 1.88 0.41 0.16 1.47 2.77 1.95 0.41 2.08

+ 0.17 + 0.68 + 0.14 -+ 0.19 -+ 0.02 -+ 0.18 + 0.14 + 0.64 -+ 0.03 -+ 0.35 + 0.28

dramatic during t h e first 24 h, whereas in the feces it continued for 48 h. Thereafter, the levels of radioactivity in all samples decreased very slowly t h r o u g h o u t the period of testing. Labeled BPD-MA initially cleared from the blood very quickly, entering the extravascular fluid c o m p a r t m e n t with a half-life of 10 - 30 min; then the clearance became slower, with a half-life of 8 h while equilibrating between the blood, extravascular fluid and the tissue.

240 10 3

E E Blood o. m

101

~ UrneFeices

~

t~

o

O -I

10 "1 0

20

40

60

80

100

Time (hours)

Fig. 3. T h e levels o f r a d i o a c t i v i t y ( m e a n + S D ) in t h e b l o o d , u r i n e a n d feces o f mice at

various times after intravenous injection of 80 pg of 3H-BPD-MA per mouse. Expressed as micrograms per gram of wet tissue or microliters per milliliter of 3H-BPD-MA. The a m o u n t of labeled compound present in the urine paralleled to some extent the level of radioactivity in the blood. Accepting 1.5 ml as the total volume of urine e x c r e t e d per mouse during 24 h [12], and assuming that the excretion was evenly spread during that period, we have calculated that 4% of the injected dose was lost through glomerular filtration during the first 24 h. The average fecal mass produced during 24 h was 1.31 g per mouse. From the calculations based on the weight of the feces and the average radioactivity of the fecal samples during various periods post injection, it appeared that more than 60% of the injected dose was cleared from the body via the intestinal tract during the first 24 h. The levels of radioactivity in the tissues of the five mice in this study, determined at 96 h post injection, were comparable with the levels determined in the tissues of normal mice (group I) tested at 96 h post injection in the study of 3H-BPD-MA biodistribution. An experiment was undertaken to determine whether counts in various tissues reflected accurately the presence of active BPD. Tumor, liver and kidney tissues were removed at 3 and 24 h after injection of 3H-BPD-MA. The drug was then extracted according to described methods. Extracted material was counted for specific radioactivity and tested for photosensitizing activity. Radioactivity and photosensitizing activity were present in all samples, and in all extracts both activities were higher at 3 h than at 24 h post injection. In all tissue extracts, except t u m o r extracts at 3 h post injection (in which 100% of counts were related to BPD cytotoxicity), the a m o u n t of BPD-MA determined by radioactivity was higher than that

241 d e ter min ed by the c y t o t o x i c i t y assay. Assuming t hat the d e t e c t e d radioactivity is representative of the total c o n t e n t of BPD-MA in tissues, the amounts o f active BPD-MA as determined by the c y t o t o x i c i t y test at 3 h post injection represent less than 40% of the total tissue SH c o n t e n t in the liver and ab o u t 70% in the kidney. At t hat time BPD -MA recovered from t u m o r was fully active. At 24 h post injection the total amounts of BPD MA recovered fr om all tissues were less than the amounts recovered at 3 h post injection. The results are summarized in Table 3. TABLE 3 Cytotoxicity of BPD--MA extracted from mouse tissues at 3 and 24 h post injection. Cytotoxicity was assessed, as previously described, on P815 target cells according to standard procedures. The level of active BPD-MA was determined using a standard curve and is expressed as the percentage of radioactive material extracted from tissues Tissue

Liver Kidney Tumor

Active BPD--MA (%) 3h

24h

37.0 71.0 100.0

7.0 13.0 39.0

4. Discussion BPD-MA appears to have several characteristics t hat could make it a suitable photosensitizer for PDT of cancer. It has an absorption peak at 692 nm; at this wavelength light penetrates deeper into tissues than the 630 nm light presently used in PDT with DHE. Moreover, it absorbs 692 nm light more efficiently than DHE absorbs 630 nm light which results in a m ore efficient activation o f the photosensitizer [ 11 ]. A good photosensitizer, in addition to absorbing tissue-penetrating light, should selectively accumulate in tumors. The results of the experiments r e p o r t e d here indicate t hat tritiated BPD-MA has the ability to localize in tumors. 3H-BPD-MA accumulates somewhat selectively in P815 and M1 tumors and even as early as 3 h after intravenous injection its levels are higher in tu m or s than in normal tissues with the except i on of the liver, spleen and kidney. Since o u r results obtained with tissue extracts suggest t hat BPD -MA is metabolized in the b o d y , the disintegrations per minute determined for each tissue at various times post injection of SH-BPD-MA may represent a m i x tu r e o f intact and metabolized drug. The radioactive BPD -MA as injected into the mice was purified, with the label attached to the molecule and fully active. Th er ef or e the injected label should distribute according to the properties of BPD-MA. The pattern of tissue distribution of 3H-BPD-MA is,

242 in general, similar to that of DHE reported by Gomer and Dougherty [13] and Dougherty [14]. The subsequent reduction of radioactivity in the tissues appears to be due to the metabolism and clearance of 3H-BPD-MA rather than to the loss of label, since the concentrations of active BPD-MA are lower than the concentrations determined by the radioactivity present in the tissues. Like DHE, 3H-BPD-MA does n o t show any real specificity for tumor tissue, since both photosensitizers reach higher concentrations in the liver, spleen and kidney than in tumors [14]. So far, the only c o m p o u n d that has shown a real specificity and has accumulated in the transplanted tumors to a much greater extent than in normal tissues is tetraphenylporphinesulfonate (TPPS) [15]. However, the use of TPPS in PDT has been precluded by its neurotoxicity [ 16, 17 ]. Although 3H-BPD-MA does n o t show specific affinity to tumors, it reaches significantly higher concentrations in the tested tumors than in many normal tissues. In general, the ratios between the t u m o r and various tissue contents of 3H-BPD-MA increase with time after injection (Table 2). From the interpretation of the cytotoxicity data obtained with the tissue extracts, it appears that the actual t u m o r to tissue ratios of active photosensitizer may be higher than those calculated on the basis of radioactivity in the tissues. Moreover, the ability to localize in tumors could be increased further by conjugating the photosensitizer to an antibody against an appropriate t u m o r marker, and using the antibody as a delivery system [18, 19]. The concentration which a photosensitizer reaches in the skin is of special interest in PDT, since the skin constitutes a barrier through which the light has to pass in order to reach a tumor. Moreover, a high content of photosensitizer in the skin in the areas exposed to ordinary daylight may cause severe phototoxic effects. This is the main side effect of PDT with HPD and DHE. The results of our experiments indicate that 3H-BPD-MA distributes between the tumors and skin favorably (Table 1) since even as early as 3 h post injection the t u m o r to skin ratio is greater than two. Preliminary experiments indicate that BPD-MA does n o t cause severe photosensitivity of the skin of animals treated with therapeutic doses, of the comp o u n d (ongoing studies). It is obvious that a photosensitizer, to be suitable for PDT, must be non-toxic at therapeutic doses. So far, we have n o t observed any signs of toxicity with BPD-MA in mice injected intravenously with four doses of 80 pg per mouse at weekly intervals. We are in the process of conducting more thorough testing. With respect to toxicity it is also important to know the fate of the c o m p o u n d in the body, including the plasma clearance and the routes of elimination. 3H-BPD-MA clears relatively quickly from the blood and well-perfused organs such as lung and heart. The rate of clearance of SH-BPD-MA from tissues seems to be similar to the rate of clearance of 3H-DHE [14]. Both compounds clear very slowly from the liver, spleen and kidney. The persistence of 3H-BPD-MA in the liver and a relatively slow clearance from

243

the intestine (Fig. 2) may suggest an enterohepatic circulation of 3H-BPDMA or its labeled metabolite. The results of the preliminary tests on the plasma clearance and the elimination of 3H-BPD-MA from the b o d y indicate that the majority of the intravenously injected dose clears from the b o d y during the first 24 h. More than 60% of the injected dose is excreted with the feces and only 4% of the dose is excreted with the urine during that time. The high radioactivity detected in some of the early gall bladder samples suggests that SH-BPD-MA is excreted from the liver to the bile and then enters the intestine with the bile. The fact that only a small percentage of the injected dose is excreted via urine may suggest that 3H-BPD-MA is present in the plasma in a proteinb o u n d form and thus escapes glomerular filtration. The pattern of elimination of 3H-BPD-MA from the body, as suggested b y our experiment with mice, appears to be similar to the pattern of elimination of hematoporphyrin from a variety of animals and man [20 - 21]. PreIiminary experiments with BPD-MA extracted from the tissues suggest that the clearance of BPD-MA from the tissues including t u m o r may be due, at least in part, to its metabolism. Since the amount of active BPD--MA appears to be reduced to about 40% at 24 h post injection from 100% at 3 h post injection, the exposure of tumors to light at 3 h rather than 24 h post injection may be more beneficial for PDT. In conclusion B P D - M A appears to be a suitable photosensitizer for PDT because of its absorption characteristics and favorable biodistribution in t u m o r and skin. Its clearance from the b o d y seems to be similar in some respects to DHE and in some respects to hematoporphyrin. The results suggest that it may be readily metabolized by the body. It requires more thorough testing in terms of toxicity and therapeutic effects before it can be introduced for use in cancer therapy. These tests are presently being carried o u t in our laboratory.

Acknowledgment This work was supported in part by a strategic grant from the Natural Sciences and Engineering Research Council of Canada.

References 1 W. Hausmann, Ueber die sensibilisierende Wirkung tierischer Farbstoffe und ihre physiologisehe Bedeutung, Biochem. Z., 14 (1908) 275 - 277. 2 A. Policard, Etudes sur les aspects offerts par les tumeurs experimentales examinees la lurni~re de Woods, C. R. Soc. Biol., 91 (1924) 1423 - 1424. 3 F. H. J. Figge, G. S. Weiland and L. O. J. Manganiello, Cancer detection and therapy: affinity of neoplastic, embryonic and traumatized tissues for porphyrins and metalloporphyrins, Proc. Soc. Exp. Biol. Med., 68 (1948) 640 - 641.

244 4 R. L. Lipson, E. J. Baldes and A. M. Olsen, The use of a derivative of hematoporphyrin in t u m o r detection, J. Natl. Cancer Inst., 26 (1961) 1 - 11. 5 T. J. Dougherty, G. B. Grindey, R. Fiel, K. R. Weishaupt and D. G. Boyle, Photoradiation therapy. II. Cure of animal tumors with hematoporphyrin and light, J. Natl. Cancerlnst., 55 (1975) 115 - 119. 6 T. J. Dougherty, Photosensitization of malignant tumors, Semin. Surg. Oncol., 2 (1986) 2 4 - 37. 7 T. J. Dougherty, Photosensitizers: therapy and detection of malignant turnouts, Photochem. Photobiol., 45 (1987) 879 - 889. 8 T. J. Dougherty, W. R. Potter and K. R. Weishaupt, The structure of the active component of hematoporphyrin derivative, in D. R. Doiron and Ch. J. Gomer (eds.), Porphyrin Location and Treatment o f Tumors, Alan R. Liss, New York, 1984, pp. 301 - 314. 9 D. Kessel, Proposed structure of the tumor-localizing fraction of HPD (hematoporphyrin derivative), Photochem. Photobiol., 44 (1986) 193 - 196. 10 D. Kessel, P. Thompson, B. Musselman and C. K. Chang, Probing the structure and stability of the tumor-localizing derivative of hematoporphyrin by reductive cleavage with LiA1H4, Cancer Res., 147 (1987) 4642 - 4645. 11 A. M. Richter, B. Kelly, J. Chow, D. J. Liu, G. H. N. Towers, D. Dolphin and J. G. Levy, Preliminary studies on a more effective phototoxic agent than hematoporphyrin, J. Natl. Cancer Inst., 79 (1987) 1327 - 1332. 12 J. K. Inglis, Introduction to Laboratory Animal Science and Technology, Pergamon Press, Oxford, 1980, p. 255. 13 C. J. Gomer and T. J. Dougherty, Determination of 3H- and 14C-hematoporphyrin derivative distribution in malignant and normal tissue, Cancer Res., 39 (1979) 146 - 151. 14 T. J. Dougherty, Photodynamic therapy (PDT) of malignant tumors, CRC Crit. Rev. Oncol. Hematol., 2 (1984) 83 - 116. 15 J. Winkelman, Jr., The distribution of tetraphenylporphinesulfonate in the tumorbearing rat, Cancer Res., 22 (1962) 589 - 596. 16 A. A. F. Sima, J. C. Kennedy, D. Blakeslee and D. M. Robertson, Experimental porphyric neuropathy: a preliminary report, Can. J. Neurol. Sci., 8 (1981) 105 - 114. 17 R. J. Riopelle and J. C. Kennedy, Some aspects of porphyrin neurotoxicity in vitro, Can. J. Physiol. Pharmacol., 60 (1982) 707 - 714. 18 D. Mew, C.-K. Wat, G. H. N. Towers and J. G. Levy, Photoimmunotherapy: treatment of animal tumors with tumor-specific monoclonal an t i b o d y - h em at o p o r p h y r i n conjugates, J. Immunol., 130 (1983) 1473 - 1477. 19 D. Mew, V. Lure, C.-K. Wat, G. H. N. Towers, C.-H. Sun, R. J. Walter, Q. Wright, M. W. Berns and J. G. Levy, Ability of specific monoclonal antibodies and conventional antisera conjugated to hematoporphyrin to label and kill selected cell lines subsequent to light activation, Cancer Res., 45 (1985) 4380 - 4386. 20 H. Smetana, Studies on photodynamic action. II. The fate of hematoporphyrin after parenteral administration. III. The influence of sensitizer on photooxidation of tissues, J. Biol. Chem., 125 (1938) 741 - 751. 21 G. C. Peck, H. P. Mack, W. A. Holbrook and F. H. J. Figge, Use of hematoporphyrin fluorescence in biliary and cancer surgery, Am. Surg., 21 (1955) 181 - 188.

Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer, in normal and tumor-bearing mice.

The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (...
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