**3.2 PDT in neurosurosurgery clinical studies – an overview**

186 Advances in the Biology, Imaging and Therapies for Glioblastoma

microscope, during resection the surgeon is able to switch between the white light mode and the fluorescent mode (blue light), where the tumour appears red fluorescent, see also

Fig. 1. Glioblastoma WHO IV at the beginning of resection in white light mode (upper row left) and under fluorescence mode (upper row right) after opening of the dura. Note the red shining PpIX fluorescence. During resection under white light conditions residual tumor (bottom row left) can be clearly detected under fluorescence mode (bottom row right).

The first results of improvement of the radicality of resection by 5-ALA fluorescence guided surgery were published in the nineties. The results of a randomised controlled multicentre phase III trial performed are summerized. The German study group investigated 322 patients enrolled by 32 investigators at 17 study centres. 161 patients were treated by fluorescence-guided surgery with 5-ALA, (20 mg/kg bodyweight; medac, Wedel, Germany), 161 were resected under conventional white light mode. In the fluorescence guided resection group tumor was resected completely in 65%, in the white light group complete tumor resection was achieved only in 36% of patients, as investigated by early postsurgical MRI. The difference between the groups was 29% [95% Confidence interval (CI) 17-40], p<0.0001. Table 1 gives a short overview about the results depending to surgery (5- ALA fluorescence guided vs. resection under white light mode).(Stummer, Pichlmeier,

Meinel, Wiestler, Zanella, & Reulen 2006)

figure 1.

Currently Kostron (2010) reviewed clinical investigations of PDT in a meta analysis, median survival of primary GBM with PDT was 22 months vs 15, in recurrent GBM 9 months vs. 3 months.(Kostron 2010) A brief overview about the relevant clinical investigations in the last decade is given by table 2. For PDT the PSs Hpd (Photofrin®) and mTHPC (Foscan) were


Table 2. Short overview - clinical investigations with PDT

Visualization and Photodynamic Therapy in Malignant Glioma - An Overview and Perspectives 189

For selective PDT it would be advantageous when the PS enriches in the malignant cells more compared to neurons or glial cells. In 2005 we investigated eight human glioma cell lines (L; LN-18,LN-229, U87MG, U373MG, D247MG, U251MG, U251MG,T98G) and twelve primary human glioma cell cultures (P) and compared the hypericin uptake with human astrocytes (AC; SV-FHAS) and cerebellar granule neurons (N) prepared from 8-day-old Sprague-Dawley rat pups (Charles River, Sulzfeld, Germany). Long term glioma cell lines and primary human glioma cell cultures showed significant higher hypericin uptake compared to neurons. Hypericin uptake in astrocytes was higher compared to glioma cells and to neurons.(Ritz et al. 2005) Another investigation done by fluorescence microscopy showed that hypericin was predominately localized in the glial envelope surrounding the neuron in a model with crayfish neuron and surrounding glia. Uzdensky et al. found a minor fraction of hypericin in the neuron compared to the glia in this model.(Uzdensky et

Predominantly perinuclear localization of hypericinis is in common concordance.(Uzdensky et al. 2001) The more detailed description of subcellular distribution of hypericin is not uniform. Due to the very short life span of the singlet-oxygen for cytotoxic reactions, the subcellular distribution of hypericin is off interest to understand PDT mechanisms in more detail. Many investigators studied in different cultured cell lines with different methods the subcellular hypericin localisation. According to our studies hypericin enriches particularly in the endoplasmatic reticulum (ER) and the Golgi apparatus (GA) in U37MG glioblastoma cells after incubation with noncytotoxic hypericin (1µM, 2h incubation time). ER is predominantly found in the perinuclear region while the GA is more distant to the nucleus,

Fig. 3a. Long-term glioblastoma, incubated with 20µM Hypericin. Note the perinuclear

**4.1 Hypericin differentiating neurons and glioma cell lines in vitro** 

al. 2003)

**4.2 Subcellular distribution of hypericin** 

see figure 3.(Ritz et al. 2007b;Ritz et al. 2008)

granular enrichment.

used, the survival time was enlarged in newly and also in patients with tumor recurrence. Side effects of PDT were modest, including skin sensitivity against sunlight and sometimes increased intracranial pressure.

Due to the small penetration depth of 5-ALA derived PpXI of 2-3mm, Eljamel et al. combined fluorescence guided resection and PDT.(Eljamel 2008) They used 5-ALA PpXI for resection and HpD for PDT. In respect to the limitation of these PSs we were encouraged to investigate a new PS combining both positive properties.
