Effect of photodynamic therapy on recurrent pituitary adenomas: clinical phase I/II trial--an early report.

Pituitary adenomas, although histologically benign, are not always curable by surgery alone, principally because of dural infiltration, as well as their peculiar anatomical location. Radiotherapy has been employed as an adjuvant therapy to address residual disease with favourable results. This approach is, however, not without side effects, and it cannot be repeated. We are therefore investigating the effectiveness of photodynamic therapy (PDT) on recurrent pituitary adenomas in humans. This study details the protocol applied to 12 patients with recurrent pituitary adenomas, which involved systemic administration of photosensitizer (Photofrin) followed, after a period of 24-48 h, by intraoperative illumination of the tumour bed using 630 nm laser light. The primary end points were visual, endocrine and radiological improvement. The incidence of side effects was also monitored. The longest follow-up is 2 years. Most patients suffering from visual acuity or field defects have shown improvement when followed for 12 months or more. Three patients showed complete recovery of their visual fields. All those who presented with functional adenomas have shown reduction in their hormone levels. Tumour volume, relative to the preoperative size, was 122, 87, 66, 60 and 46% at 4 days, and 3, 6, 18 and 24 months, respectively. One patient developed severe skin photosensitization due to early exposure to direct sunlight and three others displayed minor skin reactions. There was no treatment-related mortality or morbidity. One patient (operated transcranially) developed hemiparesis postoperatively, which recovered completely. We think this is unrelated to the treatment. This prospective study demonstrates that PDT may be safely applied to the pituitary fossa by the trans-sphenoidal route and indicates the need for a randomized, controlled trial in order to establish its therapeutic potential.

The subcellular localization of Zn(II) phthalocyanines and their redistribution on exposure to light.

The development of second-generation photosensitizers to improve photodynamic therapy (PDT) is an area of extensive research. Three such compounds that have been synthesized in our group are polysubstituted Zn(II) phthalocyanines that differ in their overall net charge (one cationic, one anionic and one neutral). The aim of this study was to characterize the drugs in terms of their uptake, cell killing efficacy and subcellular localization in RIF-1 cells in vitro and to identify any possible structure/function relationships. The results show that the relative uptakes and cell killing efficacy of each of the drugs follows the order cationic > > neutral > anionic. For the anionic and cationic drugs the initial subcellular localization was in the lysosomes as determined by fluorescence microscopy. The neutral phthalocyanine demonstrated a more diffuse localization characteristic of membrane staining with some involvement of the Golgi apparatus in the perinuclear area. Following light exposure the drugs rapidly relocalized to different sites within the cell in a manner that was apparently charge dependent and this relocalization was accompanied by an increase in the fluorescence associated with the drugs. This indicates that the primary sites of localization of these photosensitizers may not be as important as their secondary sites in producing cell killing during PDT, especially as the fluorescence intensity increases on relocalization.

Photosensitization of the endometrium with topical 5-aminolevulinic acid.

Photosensitization of the endometrium was attempted in vitro and in vivo by intrauterine administration of 5-aminolevulinic acid, which is converted to the photosensitizer protoporphyrin IX. Protoporphyrin IX was found in both functional and basal layers of the endometrial glands at concentrations nine and 10 times higher than myometrium in in vitro and in vivo experiments, respectively. Selective endometrial photosensitization is possible with topical 5-aminolevulinic acid, but it might not be distributed sufficiently evenly for use as a sensitizing agent in photodynamic ablation.

The quantitative determination of Photofrin and Polyhaematoporphyrin in plasma: pitfalls and inaccuracies.

An accurate and relatively rapid fluorometric assay for Photofrin, or our own preparation Polyhaematoporphyrin (PHP), in plasma has been developed. This method takes into account the significant proportion of hydrolysis-resistant material now known to be present in these sensitizers, which has undoubtedly led to the inaccurate assessment of these drugs and other preparations of haematoporphyrin derivative (HPD) in numerous studies. The method was devised to allow for incomplete hydrolysis by calibration with plasma samples to which known amounts of photosensitizer were added in vitro. It was then compared with an "absolute" method in which plasma from animals that had received 14C-labelled drug was subjected to radioactivity assay. The two approaches gave almost identical results. The calibration method is applicable to the determination of any HPD-derived drug from patient or animal studies. As an example of its use in the present study, it was applied to the determination of the pharmacokinetics of PHP in the rat.

The biosynthesis of the chromophore of phycocyanin. Pathway of reduction of biliverdin to phycocyanobilin.

The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin.

Ferrochelatase activity in the photosynthetic alga Cyanidium caldarium. Development of the enzyme during biosynthesis of photosynthetic pigments.

Dark-grown cells of the photosynthetic alga Cyanidium caldarium were shown to contain ferrochelatase activity, which increased markedly when the cells were induced to form pigments by exposure to light. Km values for the crude enzyme preparation were 14.8 microM and 6.5 microM for binding of Co2+ and deuteroporphyrin IX respectively.

Biosynthesis of phycobiliproteins. Incorporation of biliverdin into phycocyanin of the red alga Cyanidium caldarium.

14C-labelled biliverdin IX alpha was administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Between 9 and 12% of the phycobiliprotein chromophore produced in such cultures was derived from exogenous biliverdin. These results demonstrate that biliverdin is an intermediate in the biosynthesis of phycobiliproteins.

Biosynthesis of the chromophore of phycobiliproteins. A study of mesohaem and mesobiliverdin as possible intermediates and further evidence for an algal haem oxygenase.

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.

Incorporation of haem into phycocyanobilin in levulinic acid treated Cyanidium caldarium.

Cells of the red alga Cyanidium caldarium were preincubated with 5 mmol 1(-1) levulinic acid (LA) and subsequently incubated with (14)C-labelled haem (5.67 Bq nmol(-1)). Phycocyanin was isolated. The specific radio-activity of its chromophore phycocyanobilin (PCB) was determined after cleavage and purification by thin layer chromatography. The percentage of PCB formed from labelled haem within 0.5 h was considerably higher in LA treated cells than in non-treated controls. This difference disappeared after prolonged incubation (16.5 h) with haem. The results are interpreted as possible incorporation of haem into preexisting apoprotein.

The effect of N-methylprotoporphyrin IX on the synthesis of photosynthetic pigments in Cyanidium caldarium. Further evidence for the role of haem in the biosynthesis of plant billins.

N-Methylprotoporphyrin IX strongly inhibits synthesis of phycocyanobilin, but not chlorophyll a, in the dark. In the light, both phycocyanin and chlorophyll a synthesis are inhibited in parallel. These results are consistent with the intermediacy of haem in algal bilin synthesis and suggest a control mechanism for chlorophyll a synthesis, previously unknown.

A study of the mechanism of quercetin oxygenation by 18O labelling. A comparison of the mechanism with that of haem degradation.

The mechanism of quercetin oxygenation, which is formally analogous to haem degradation, was studied by using 18O labelling. In both the enzymic oxygenation (catalysed by quercetinase) and the non-enzymic reaction (base-catalysed), both oxygen atoms incorporated into product were derived from a single oxygen molecule. Quercetin oxygenation therefore occurs by a classical dioxygenase mechanism and is not an appropriate model for study of the mechanism of haem catabolism.

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.

The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.

The cross-linking of rabbit skeletal muscle sarcoplasmic reticulum protein.

Sarcoplasmic reticulum proteins have been cross-linked in situ with two reagents, the disulphide-bridged bifunctional imido ester, dimethyl-3,3'-dithiobispropionimidate dihydrochloride and the mild oxidant cupric phenanthroline. Analysis of proteins so cross-linked by electrophoresis on agarose/acrylamide gels reveals that a series of new polypeptides, up to a molecular weight of 900 000, are formed. These have molecular weights which are multiples of 100 000. Further analysis of samples by electrophoresis in a second dimensions containing a reducing agent revealed the monomeric polypeptides from which the cross-linked polypeptides were formed. With dimethyl 3,3'-dithiobispropionimidate dihydrochloride homopolymers of the Ca2+-stimulated ATPase, calsequestrin and/or calcium binding protein were formed. With cupric phenanthroline only the Ca2+-stimulated ATPase was involved in polymer formation. It has been confirmed on another gel system that these two proteins which are involved in Ca2+ binding are not cross-linked intermolecularly with this latter reagent. We conclude that the 100 000 dalton Ca2+-stimulated ATPase polypeptides are within 2 A of each other in the membrane while calsequestrin and/or calcium binding protein are within 11 A of each other. Although there appears to be no limit to the extent of cross-linking of any of these polypeptides there is not indication of heteropolymer associations between them.