In vivo biological activity of the components of haematoporphyrin derivative

Abstract

Summary.-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.

The sensitivity of normal brain and intracranially implanted VX2 tumour to interstitial photodynamic therapy

“…The model (Patterson et al, 1990) was derived from the observation of a sharp boundary that usually occurs between PDT-induced tissue necrosis and undamaged adjacent tissue (Berenbaum et al, 1982;Potter, 1989). The model states that, for a given tissue and photosensitiser combination, necrosis will occur if the number of photons absorbed by the photosensitiser per unit tissue volume exceeds a threshold value (1).…”

Retention and photodynamic effects of haematoporphyrin derivative in cells after prolonged cultivation in the presence of porphyrin

“…after alkali treatment, see Materials and methods) contains at least 4 defined porphyrins (haematoporphyrin (Hp), two isomers of monohydroxy-monovinyldeuteroporphyrin (HvD) and protoporphyrin (PP)) (Bonnett et al, 1980;Moan & Sommer 1981). In addition to these compounds, a certain amount of unknown material is found, somewhat dependent on the method used to separate the components (Moan et al, , 1982aBerenbaum et al, 1982;Dougherty et al, 1983). A large fraction of these unknown components are aggregates, some of which may be composed of porphyrins bound strongly together with, for instance, covalent bonds (Moan et al, 1982a, Berenbaum et al, 1982.…”

“…In addition to these compounds, a certain amount of unknown material is found, somewhat dependent on the method used to separate the components (Moan et al, , 1982aBerenbaum et al, 1982;Dougherty et al, 1983). A large fraction of these unknown components are aggregates, some of which may be composed of porphyrins bound strongly together with, for instance, covalent bonds (Moan et al, 1982a, Berenbaum et al, 1982. This conclusion is supported by the present findings that the 360-370nm absorption, characteristic of aggregated porphyrins, is still present at low concentration and in a variety of solvents.…”

In vitro photosensitization of tumour cell enzymes by Photofrin II administered in vivo

“…The more hydrophilic porphyrin species of HpD, such as the isomers of haematoporphyrin and hydroxyethylvinyldeuteroporphyrin, were probably present in sufficient levels in the cytosol soon after injection such that exposure to light caused oxidative damage to cytosolic proteins. On the other hand, Photofrin II, a mixture of porphyrins enriched in the hydrophobic species (80-90% as reported by Dougherty (1987)), presumably dihaematoporphyrin ethers and/or esters (Berenbaum et al, 1982;Byrne et al, 1987;Dougherty, 1987;Kessel et al, 1987), would be expected to accumulate primarily in the more hydrophobic regions, such as cell membranes. If this were the case, the lower concentration of hydrophilic components (20%) in Photofrin II would be less able to produce sufficient 102 in the cytosol to cause inhibition of pyruvate kinase, the result we observed.…”