Biological and biophysical properties of the tumor-localizing component of hematoporphyrin derivative

Abstract

Reverse-phase chromatography, aqueous gel exclusion, and nonaqueous gel exclusion were assessed as procedures for preparative fractionation of the tumor-localizing product hematoporphyrin derivative. Porphyrin accumulation, fluorescence, and photodynamic cytotoxicity were monitored using the murine Sarcoma 180 tumor. Aqueous gel exclusion chromatography can provide a hematoporphyrin derivative fraction enriched in the tumor-localizing component. A further enrichment occurs when this procedure is carried out at 55 degrees C, but nonlocalizing porphyrins could not be eliminated. While providing a better separation, reverse-phase chromatography cannot provide a tumor-localizing fraction free from contaminating protoporphyrin. However, this and other contaminants can be eliminated from the tumor-localizing fraction via nonaqueous gel exclusion chromatography. This latter separation provides two tumor-localizing products: a fast-eluting fraction enriched in the major photosensitizing component(s); and a more complex slowly eluting fraction enriched in fluorescence localizers.

Chart 1

Reverse-phase HPLC analysis of HPD.

Chart 2

HPLC analysis of the first-eluted HPD fractions from a P-10 column. A, solvent, water at 20°C; B, solvent, water at 55°C; C, solvent, formamide:water (1:1) at 20°C.

Chart 3

Preparative fractionation of HPD on an LH-20 column. Absorbance at 500 nm was monitored; specified fractions were pooled for HPLC analysis.

Chart 4

HPLC analysis of pooled HPD fractions eluted from an LH-20 column (Chart 3). A, Fractions 1 to 3; B, Fractions 4 and 5; C, Fractions 6 to 10.

Chart 5

Relative elution volume versus molecular weight of a series of dyes on an analytical LH-20 column. 1, neutral red; 2, methylene blue; 3, phenol red; 4, merocyanine 530; 5, hematoporphyrin; 6, eosin Y; 7, rose bengal; 8, Fraction B (Chart 4); 9, Fraction A (Chart 4); 10, vitamin B₁₂.

Chart 6

Degradation of Fraction A (Chart 4) when held at (left) 100°C for 100 min or (right) 4°C for 100 days.

Chart 7

Absorption patterns of 1-μg/ml porphyrin solutions in water, methanol (MeOH), or 10 mm aqueous CTAB. Left, Fraction A (Chart 4); right, HP. For the latter compound, the tracings in CTAB and methanol were identical.