In vivo biological activity of the components of haematoporphyrin derivative

The in vivo biological activity of various fractions and components of haematoporphyrin derivative (HpD) have been determined by measuring the depth of necrosis of implanted tumours in mice exposed to light after the administration of standard doses of porphyrins dissolved in alkali. In this assay, haematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin are inactive, but the mono- and di-acetates of haematoporphyrin (which are major components of HpD) and acetoxyethylvinyldeuteroporphyrin are active. However, the situation appears to be more complex than this. The normal method for preparing HpD for injection involves an alkali treatment which causes hydrolysis and elimination of the acetoxy functions, and the only recognized products (haematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin) are inactive in the in vivo assay. It is concluded that the active component here is a porphyrin, possibly a dimer or oligomer, which is retained on the column during the normal separation by HPLC. This conclusion is supported by the observations that (i) the crude material obtained from the spent column is active without further alkali treatment, and (ii) activity develops over 30 min, when HpD or the mono- or diacetates of haematoporphyrin are treated with sodium bicarbonate in aqueous DMSO. The advantages of working with a pure substance (e.g. haematoporphyrin diacetate) rather than a mixture (HpD) are stressed.