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The distribution of isotopically labeled hematoporphyrin derivative (HPD) has been studied in mice bearing the spontaneous mammary tumor (fast growing). In stomach, liver, spleen, and pancreas, 3 hr after i.p. injection of [3H]HPD, grains were uniformly distributed over the tissue sections. After 24 hr, the grain density overlying parenchymous areas of these tissues was lower than that over the stromal or reticuloendothelial areas. In the spontaneous mammary tumor (fast growing), higher grain densities were seen over pseudocapsule, stromal septa, and necrotic areas at 3, 6, 12, 24, and 48 hr after injection. At 168 hr postinjection, only isolated stomal cells, presumably macrophages, showed high grain densities. From the temporal changes observed in the distributions of HPD in normal tissues and the relative stability of the distribution seen in the spontaneous mammary tumor (fast growing), we speculate that tissue factors such as vascular permeability, lack of an adequate lymphatic drainage, and nonspecific binding of serum proteins to stromal elements may be responsible for or contribute to the preferential uptake and/or retention of HPD observed in both human and animal tumors.

Hematoporphyrin and white light exerted a lethal effect on two cell lines of retinoblastoma cells. The lowest values of dye concentration and light exposure capable of killing an entire cell population were, respectively, 2 X 10(-5) M and 6 min exposure to an irradiance of 6.0 microwatts/sq mm. Cells exposed to light in the presence of the dye did not require lengthy incubation periods but were rapidly killed with increasing periods of light exposure. Cells washed free of the dye, however, required a minimum sensitizing period of 3.5 hr to achieve a value close to 100% cell death. An inhibitor of this photodynamic process was demonstrated in normal serum. When the concentration of either fetal calf serum, rabbit serum, or rabbit plasma was increased to 25% from a standard 10%, there was as much as a 100-fold greater requirement of hematoporphyrin concentration to produce the same lethal response. The suggested explanation for this phenomenon is a porphyrin-binding plasma factor, hemopexin, which is a natural beta-glycoprotein believed to be responsible for the transport of circulating porphyrins to the liver for their elimination. Retinoblastoma cells grow in suspension and thereby provide an excellent tool for study of photosensitive dyes, especially in the case of the rapidly growing Y79 cell line.

A variety of cell lines have been prepared by fusion of the murine WEH1 3B cell line with peripheral blood leukocytes from a patient with chronic granulocytic leukemia. Fusion products were selected for their ability to produce a leukemia-associated antigen (CAMAL) previously described. One such line which originally produced CAMAL subsequently lost this ability and was used as a negative control. A number of antibodies were conjugated to hematoporphyrin (HP) and tested for their ability to bind to cell lines as detected by either fluorescence or by their ability to kill cells after light activation. The antibodies used were: rabbit anti-Hu (a conventional rabbit antiserum raised to membrane preparations from normal human peripheral blood leukocytes which served as a positive control); CAMAL-1 (a monoclonal gamma 1 antibody with specificity for the CAMAL antigen); and L1210 (an irrelevant monoclonal gamma 1 antibody). HP was conjugated to the antibodies by a carbodiimide procedure. When labeled cells were examined by fluorescence microscopy, it was apparent that both the rabbit antibody and CAMAL-1:HP showed positive labeling. The ability of the antibody:HP conjugates to kill labeled cells following light activation was tested. It was shown that rabbit anti-Hu:HP and CAMAL-1:HP conjugates were capable of killing significant numbers of cells when HP concentrations were as low as 1.2 ng/10(6) cells, whereas similarly treated cells exposed to either L1210:HP or HP alone did not exhibit significant killing until concentrations reached 240 and 120 ng/10(6) cells, respectively. Further experiments in which other cell lines were tested, all at HP concentrations of 12 ng/10(6) cells, demonstrated that those lines producing CAMAL were killed, whereas negative lines were not.

Administration of hematoporphyrin derivative i.v. followed by local exposure to red light has resulted in complete or partial response in 111 of 113 cutaneous or s.c. malignant lesions. Tumors treated have included carcinomas of the breast, colon, prostate, squamous cell, basal cell, and endometrium; malignant melanoma; mycosis fungoides; chondrosarcoma; and angiosarcoma. No type has been found to be unresponsive. In several cases complete clearing of chest wall metastatis has been achieved in treated areas. Deep-seated and pigmented tumors required a higher dose of drug for effective treatment than did the more superficial and nonpigmented lesions. A high therapeutic ratio between tumor and skin response has been obtained by allowing at least 3 days between drug injection and exposure to the therapeutic light for 2,5-mg/kg doses and at least a 4-day interval for 5.0-mg/kg doses.

Hematoporphyrin derivative (HPD), a complex mixture of porphyrins derived from hematoporphyrin, has been used for localization and photoradiation therapy of tumors. In this report, we describe characterization of HPD and of its component porphyrins by reverse-phase thin-layer chromatography. Uptake of major HPD components by leukemia L1210 cells in vitro and by the Sarcoma 180 tumor in vivo were also examined. These data suggest that the apparent photosensitization of intact cells mediated by hematoporphyrin was associated with the uptake of more hydrophobic porphyrins present as impurities. In the Sarcoma 180 tumor, the fluorescent porphyrin persisting for two days in vivo after HPD administration was found to migrate with hematoporphyrin in a reverse-phase thin-layer chromatography system. Our results suggest that hematoporphyrin may be unable to cross the cell membrane readily in either direction and that long-persisting fluorescence resulting from exposure of tumor tissues to HPD results from transformation of a membrane-permeable HPD component to hematoporphyrin.