**2. History of PDT**

The newer history of PDT starts with the observations of Von Tappeiner and Raab at the Maximilian Ludwig University in Munich. In 1900, Raab first reported on the chemical sensitisation of tissue by light.(Raab 1900) Von Tappeiner described in 1904 the so called "photodynamic reaction".(Tappeiner & Jodlbauer 1904) He believed that this effect was based on fluorescence. In contrast Neiser (Breslau) and Dreyer (Finsen Institue in Kopenhagen) described a sensitisation by light for photodynamic reaction.(Dreyer 1903;Neisser & Halberstaedtter 1904) At this time Ledoux-Lebards already proved the concept of the presence of oxygen as a condition for PDT at the Institute Pasteur in Paris (1902).(Ledoux-Lebards 1902) In this era skin diseases were treated with chinidin, acridin and eosin with unsatisfying results.

Already from the beginning of PDT, haematoporphyrin (Hp) was of special interest. Hausmann used Hp for photodynamic investigations in mice in 1911.(Hausmann 1911) In 1913, Meyer-Betz studied Hp to determine its biological effects on himself. After exposition to sunlight he suffered from extensive phototoxic reactions.(Meyer-Betz 1913) Policard detected 1924 in rat sarcoma a red fluorescence after Hp administration.(Policard 1924) In 1942 Auler and Banzer reported on the affinity of neoplastic tissues for Hp in tumor, metastases and lymphatic vessels in patients suffering cancer.(Auler & Banzer 1942) Further investigations were performed by Figge et al. in 1948; they demonstrated the properties of Hp to localize tumors.(Figge, Weiland, & Manganiello 1948) Due to high toxic reactions of

Visualization and Photodynamic Therapy in Malignant Glioma - An Overview and Perspectives 185

visualisation.(Stummer et al. 2000;Stummer et al. 2006;Stummer et al. 2008) 5-ALA is a precursor, converted in malignant cells to PpIX, the fluorescent substance. 5-ALA is the first substrate in the heme biosynthesis. Heme biosynthesis consists of eight discrete enzymes catalysed steps which involve the mitochondrial (the first and the last three steps) and cytosolic (the other four intermediate steps) compartments of the cell. In the first step 5-ALA is produced by ALA synthetase in the mitochondria. This is the rate-limiting step in heme biosynthesis. 5-ALA is actively transported to the cytoplasm. After reentry into the mitochondrion, PpIX is produced. PpIX is the last step in the heme pathway before forming heme by insertion of ferrous iron by the enzyme ferrochelatase. Mitochondrial ferrochelatase is dependent on mitochondrial energy generation. In malignant tissue ferrochelatase is reduced, therefore PpIX, a strongly fluorescent and effective tissue PS accumulates in higher concentrations after application of 5-ALA in gliomas.(Kemmner et al. 2008) Some more reasons for higher accumulation of PSs should be mentioned. On the surface of tumour cells more low-density lipoprotein (LDL) receptors are found than on the surface of normal cells.(Maziere, Morliere, & Santus 1991) Increased porphobilinogendeaminase activity in malignant glioma cells also leads to higher PpIX concentration. (Berkovitch-Luria et al. 2011;Greenbaum et al. 2002) At least slightly elevated

temperature increases also the rate of biosynthesis of PpIX.

**3. Photodiagnosis and photodynamic therapy in neurosurgery** 

glioma treatment method. This topic will be discussed later in the chapter.

HpD and mTHPC are currently in use.

**3.1 Photodiagnosis (PD) and PDT with 5-ALA PpIX** 

Currently, standard treatment of glioblastoma is based on microsurgical tumour resection, radiation and chemotherapy. Overall prognosis of glioblastoma patients remains poor; therefore, new therapeutical options are necessary. Glioblastomas are diffuse infiltrating tumors, with growth patterns according to Scherer as follows: (i)perineuronal growth (perineuronal satellitosis); (ii) surface (subpial) growth; (iii) perivascular growth and (iv) intrafascicular growth.(Peiffer & Kleihues 1999) Due to the fact that tumor recurrence occurs most frequently at the resection margins, PDT of malignant glioma might be a promising treatment option as a local therapy. Additionally PDT might be able to reach the so called Guerilla cells(Claes, Idema, & Wesseling 2007), tumor cells localized in the brain adjacent to tumor region (BAT region) by local therapy at the end of the resection and perhaps by PDT stimulated anti-tumor immunity. Stimulation of anti-tumor immunity by PDT is off increasing relevance, an opportunity for PDT to become quite more than another local

In the 1970s, when lasers and optical light delivery systems became available, the therapeutic use of Hp maintained interest also in neurosurgery. In 1972, Diamond et al. studied the photodynamic effects of Hp in glioma cell cultures where addition of 10*-*5 M Hp and exposure to light caused cell death.(Diamond et al. 1972) In 1975 the same group investigated the photodynamic effect of Hp *in vivo* in Fisher rats and subcutaneous implanted glioma cells; the authors demonstrated a time dependent cell death of glioma cells increasing by time of light exposure. For clinical use of PDT in Neurosurgery mainly

Introduction of 5-ALA fluorescence guided resection was a milestone in neurosurgery in the last fifteen years. Great contribution was done by Stummer and coworkers. 5-ALA is orally applied about 4 hours before surgery. Surgery is performed under operating

Hp, in 1955 a hematoporphyrin derivat (HpD) was developed by Schwartz et al..(Schwartz, Absolon, & Vermund 1955) This derivat also contained many components of hematoporphyrins. Lipson et al. used the HpD *in vivo* and in patients for tumor detection and localisation in the early sixties.(Lipson, Baldes, & Olsen 1964)

A milestone in PDT was done by Dougherty in 1973.(Dougherty 1973) Dougherty postulated the criteria for PSs and for PDT. Essential for a successful PS is less or no toxicity without light, selective enrichment in the tumor or affected tissue and activation by light with a wavelength of 600 nm or more.(Dougherty et al. 1978;Dougherty et al. 1998a) HpD was further purified by Dougherty's group to Photofrin®. Photofrin is up to now for PDT drug approved.

## **2.1 First and second generation photosensitizers**

HpD and Photofrin® are first generation PSs. The maximum of absorption of HpD is 628 to 632 nm. Penetration depth is about 5 mm. *In vivo* the concentration of HpD is twelve times higher compared to normal brain tissue. In clinical investigations the concentration was 1:2.5 to 1:4 fold. (Kostron, Obwegeser, & Jakober 1996) This first generation PSs have some disadvantages, e.g. high impurity, prolonged skin photosensitivity about several weeks and low absorbance at 630 nm, where tissue penetration of light is low. To improve this, second generation photosensitizers (phthalocyanines, naphthalocyanins, benzoporphyrins, chlorines, purpurins, texaphyrins, porphycenes, pheophobides, bacteriochlorins, etc.) were introduced (Juzeniene, Peng, & Moan 2007). Second generation PSs have a high absorbance in the region of 650-850 nm and produce adequate singlet oxygen. Meta-tetra hydroxyphenylchlorin (m-THPC; Foscan®, Biolitec AG) and benzoporphyrin derivative monoacid A (BPD-MA; Visudyne®, QLT Inc. and Novartis Opthalmics) are approved drugs for clinical use. The second generation PS mTHPC has its maximum absorption at 652 nm. Phototoxic reaction had been observed up to a depth of 15 mm. For metatetrahydroxyphenylchlorin (mTHPC) a ratio tumor to normal tissue of more than 80:1 has been described *in vivo* after implantation of C6 glioma in Spraque-Dawley rats. In clinical applications the ratio tumor to normal brain tissue was 20:1.(Dougherty, Gomer, Henderson, Jori, Kessel, Korbelik, Moan, & Peng 1998a;Obwegeser, Jakober, & Kostron 1998) Several more PSs are available, but have been less usage in neurosurgery. Third generation PSs are second generation photosensitizers bound to carriers for selective accumulation in the tumor.

#### **2.2 Prodrug: 5-aminolevulinic acid derived protoporphyrin IX**

5-aminolevulinic acid (5-ALA) a prodrug transformed to 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA PpIX) is especially used for photodiagnosis (PD) although properties for as a PS are known. In 1955 Scott described the transitory hypersensitivity to sunlight following exogenous administration of 5-ALA.(Scott 1955) First description about the use of 5-ALA as a porphyrin precursor in PDT was done by Malik and Lugaci, who demonstrated, that exogenous 5-ALA PpIX in combination with light led to inactivation of leukemic cells.(Malik & Lugaci 1987) Kennedy et al. reported about successful treatment of malignant and precancerous skin diseases in 1990.(Kennedy, Pottier, & Pross 1990) The use of 5-ALA PpIX in neurosurgery for fluorescence guided resection of glioblastoma was a milestone. Stummer et al. demonstrated convincingly that the radicality of tumor resection and thus the outcome of patients improves significantly by intraoperative tumor

Hp, in 1955 a hematoporphyrin derivat (HpD) was developed by Schwartz et al..(Schwartz, Absolon, & Vermund 1955) This derivat also contained many components of hematoporphyrins. Lipson et al. used the HpD *in vivo* and in patients for tumor detection

A milestone in PDT was done by Dougherty in 1973.(Dougherty 1973) Dougherty postulated the criteria for PSs and for PDT. Essential for a successful PS is less or no toxicity without light, selective enrichment in the tumor or affected tissue and activation by light with a wavelength of 600 nm or more.(Dougherty et al. 1978;Dougherty et al. 1998a) HpD was further purified by Dougherty's group to Photofrin®. Photofrin is up to now for PDT

HpD and Photofrin® are first generation PSs. The maximum of absorption of HpD is 628 to 632 nm. Penetration depth is about 5 mm. *In vivo* the concentration of HpD is twelve times higher compared to normal brain tissue. In clinical investigations the concentration was 1:2.5 to 1:4 fold. (Kostron, Obwegeser, & Jakober 1996) This first generation PSs have some disadvantages, e.g. high impurity, prolonged skin photosensitivity about several weeks and low absorbance at 630 nm, where tissue penetration of light is low. To improve this, second generation photosensitizers (phthalocyanines, naphthalocyanins, benzoporphyrins, chlorines, purpurins, texaphyrins, porphycenes, pheophobides, bacteriochlorins, etc.) were introduced (Juzeniene, Peng, & Moan 2007). Second generation PSs have a high absorbance in the region of 650-850 nm and produce adequate singlet oxygen. Meta-tetra hydroxyphenylchlorin (m-THPC; Foscan®, Biolitec AG) and benzoporphyrin derivative monoacid A (BPD-MA; Visudyne®, QLT Inc. and Novartis Opthalmics) are approved drugs for clinical use. The second generation PS mTHPC has its maximum absorption at 652 nm. Phototoxic reaction had been observed up to a depth of 15 mm. For metatetrahydroxyphenylchlorin (mTHPC) a ratio tumor to normal tissue of more than 80:1 has been described *in vivo* after implantation of C6 glioma in Spraque-Dawley rats. In clinical applications the ratio tumor to normal brain tissue was 20:1.(Dougherty, Gomer, Henderson, Jori, Kessel, Korbelik, Moan, & Peng 1998a;Obwegeser, Jakober, & Kostron 1998) Several more PSs are available, but have been less usage in neurosurgery. Third generation PSs are second generation photosensitizers bound to carriers for selective accumulation in the

5-aminolevulinic acid (5-ALA) a prodrug transformed to 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA PpIX) is especially used for photodiagnosis (PD) although properties for as a PS are known. In 1955 Scott described the transitory hypersensitivity to sunlight following exogenous administration of 5-ALA.(Scott 1955) First description about the use of 5-ALA as a porphyrin precursor in PDT was done by Malik and Lugaci, who demonstrated, that exogenous 5-ALA PpIX in combination with light led to inactivation of leukemic cells.(Malik & Lugaci 1987) Kennedy et al. reported about successful treatment of malignant and precancerous skin diseases in 1990.(Kennedy, Pottier, & Pross 1990) The use of 5-ALA PpIX in neurosurgery for fluorescence guided resection of glioblastoma was a milestone. Stummer et al. demonstrated convincingly that the radicality of tumor resection and thus the outcome of patients improves significantly by intraoperative tumor

and localisation in the early sixties.(Lipson, Baldes, & Olsen 1964)

**2.2 Prodrug: 5-aminolevulinic acid derived protoporphyrin IX** 

**2.1 First and second generation photosensitizers** 

drug approved.

tumor.

visualisation.(Stummer et al. 2000;Stummer et al. 2006;Stummer et al. 2008) 5-ALA is a precursor, converted in malignant cells to PpIX, the fluorescent substance. 5-ALA is the first substrate in the heme biosynthesis. Heme biosynthesis consists of eight discrete enzymes catalysed steps which involve the mitochondrial (the first and the last three steps) and cytosolic (the other four intermediate steps) compartments of the cell. In the first step 5-ALA is produced by ALA synthetase in the mitochondria. This is the rate-limiting step in heme biosynthesis. 5-ALA is actively transported to the cytoplasm. After reentry into the mitochondrion, PpIX is produced. PpIX is the last step in the heme pathway before forming heme by insertion of ferrous iron by the enzyme ferrochelatase. Mitochondrial ferrochelatase is dependent on mitochondrial energy generation. In malignant tissue ferrochelatase is reduced, therefore PpIX, a strongly fluorescent and effective tissue PS accumulates in higher concentrations after application of 5-ALA in gliomas.(Kemmner et al. 2008) Some more reasons for higher accumulation of PSs should be mentioned. On the surface of tumour cells more low-density lipoprotein (LDL) receptors are found than on the surface of normal cells.(Maziere, Morliere, & Santus 1991) Increased porphobilinogendeaminase activity in malignant glioma cells also leads to higher PpIX concentration. (Berkovitch-Luria et al. 2011;Greenbaum et al. 2002) At least slightly elevated temperature increases also the rate of biosynthesis of PpIX.
