Several teams have applied the concept of compartmental targeting to enhance photodynamic efficacy16-21
. Zhou et al
noticed the importance of the choice of DLI to obtain a good treatment efficacy together with a maximal protection of normal tissues. Many subsequent investigations commented on the impact of DLI on the photodynamic effect, with significant improvement of efficacy with shorter time intervals, corresponding to high plasma levels of the drug23-25
. Veenhuizen et al.
investigated the impact of a double injection of 0.3 mg/kg mTHPC at 1-3 and 48h before illumination6
. Although mTHPC levels in the tumour were the highest at the longer DLIs, response was better at shorter time intervals and high plasma levels. The combination of 2 injections with the aim to obtain high drug levels in both tumour and vascular compartments did not significantly improve results. Identical observations were made by the same group when both injections were separated by a time interval of 72h, despite the fact that the total drug dose was doubled4
. Using a so-called vascular photosensitizer, Dolmans et al
. demonstrated that a double injection of this drug at 15 minutes and 4h before illumination was significantly better than the single drug dose at any of those time points5
. They attributed this positive effect to the fact that fluorescence studies indicated a more homogeneous staining of both endothelial and perivascular structures following a double injection.
In a previous study, we used high resolution confocal fluorescence imaging to simultaneously map microscopic intratumoral mTHPC localization with respect to perfused vasculature as a function of the time after injection9
. A progressive gradient of PS fluorescence was observed from the lumen, to endothelial cells, parenchyma adjacent to the vessels, and finally tumour cells remote from the vascular structures. Three hours post injection, maximal mTHPC fluorescence was detected in the periluminal structures (within 15 μm). After 24h, fluorescence was about three times higher 140 μm from the vessel, corresponding to parenchyma localisation. We therefore chose those two time points to evaluate the impact of fractionated mTHPC delivery on PDT-induced regional distribution of apoptosis and overall tumour regrowth.
With regard to the tumour regrowth curves (), it appears that irrespective of the single drug dose used, longer time intervals (24h) are less effective, producing < 20% tumour cure. These results are in agreement with previous mTHPC studies4-6
. A 100% cure rate was observed when two separate injections of 0.15 mg/kg were administered 3 and 24h before illumination (). A positive impact of drug fractionation has never been mentioned previously for mTHPC-PDT. This should probably be attributed to the time points that were chosen in the earlier studies, 48 and 72h4, 6
. Indeed from our intratumor drug distribution studies, it appears that at those longer time points, maximal mTHPC fluorescence within the tumour is extremely remote from the vessels, thus indicating drug accumulation in regions that are potentially hypoxic and result in reduced PDT efficacy9
Irrespective of DLI, necrosis strongly contributes to cell death as observed through HES staining (). This result is anticipated considering that mTHPC is a strong mediator of photoinduced necrosis26
. Regional distribution of apoptotic features induced by PDT has been poorly documented. An immunohistochemistry study by Engbrecht et al
. demonstrated that Photofrin® mediated PDT induced apoptosis firstly in the endothelial cells bordering occluded blood vessels and became more widespread at later time points14
. This observation relied on a double fluorescent staining of endothelial cells (anti-PECAM: Platelet Endothelial Cell Adhesion Molecule) and apoptosis via the TUNEL technique (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling). A more recent study of Henderson et al10
investigated tumour grafts treated by 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH)-PDT with high and low fluences and fluence rates (48 and 128 J/cm2
, 14 and 112 mW/cm2
). They demonstrated that extent and regional distribution of tumour and vascular damage, particularly apoptosis, correlated with fluence and fluence rate used during treatment.
To enable a new approach for simultaneous co-labelling of vessels and apoptosis in the same section, cleaved caspase-3 is targeted for apoptosis and murine collagen IV is used to mark basal membranes of vessels. The advantage of this new method is that, on the same sample, apoptosis and vascular structures can be observed along with classical morphopathological features. When the PDT effect is extremely poor (24h), vessels are intact and only parenchyma cells show apoptotic features (). Intermediate cure rates obtained with DLIs of 3 and 6h mostly indicate vascular destruction, since collagen IV staining is absent along with a very limited staining of cleaved caspase-3 (). A fractionated injection results in vascular effects with a poor collagen IV staining, together with massive apoptosis within the bulk tumour ().
Historically, PDT protocols were established on the assumption that illumination should be performed at the highest tumour-to-normal drug ratios in order to protect healthy surrounding tissue and achieve a good tumouricidal effect27
. These ratios are only observed at long DLIs. Recent preclinical work however suggests that better cure rates could be obtained at shorter time intervals, corresponding to high plasma levels of the drug8
. From our work, it appears that the highest efficacy can be obtained when combining both propositions, aiming at the simultaneous destruction of tumour vasculature and neoplastic cells. The time point chosen for drug administration and illumination appears to be critical, since our study is the first to observe a benefit from mTHPC fractionation. It may be hypothesized that this is a consequence of the double injection resulting in a more homogeneous distribution of the drug in vascular structures as well as in tumour cells close to and more distant from vessels. These time points should not be determined solely on the basis of the plasma and bulk tumour drug levels obtained by extraction but rather by the temporal-spatial distribution of the photosensitizer within the tumour.
Although the excellent PDT efficacy obtained from dose fractionation at 24h and 3h may be unique to mTHPC due to its tight cell/tissue binding properties, the tumour response results clearly demonstrate the importance of DLI informed by detailed drug distribution studies like those performed for mTHPC9, 28