**4. In vivo assessment of therapies**

In addition to using microdialysis to evaluate the baseline concentration of molecules involved glioma pathogenesis, several studies have also made use of the technique to investigate the response to treatment with chemo- and radiotherapy.

#### **4.1 Chemotherapy**

Microdialysis may be used to evaluate both chemotherapeutic pharmacokinetics and pharmacodynamics. Interestingly, the earliest example of clinical microdialysis in patients with brain tumours to investigate a drug's pharmacology focused not on chemotherapy but on the antimicrobial rifampicin. Mindermann et al recruited 5 patients with HGG and 3 patients with LGG (Mindermann 1999). All patients received a single pre-operative dose of 600mg rifampicin 3 hours before skin incision. Patients then underwent craniotomy and tumour resection with a single microdialysis catheter placed distantly from the resection margin in grossly unaffected brain around tumour (BAT). A LWCO catheter was infused

Clinical Microdialysis in Glioma 155

experiment determined a mean BPA recovery of 66.8 ± 8.8%. The group also monitored glucose and its metabolites, glutamate and glycerol throughout the procedure. Four patients with WHO grade IV glioblastoma multiforme (GBM) were recruited. One patient underwent gross total resection, one a subtotal resection, and two stereotactic biopsies. In the patients that underwent resection a microdialysis catheter was placed within 5mm of the resection margin (T), and in the patients undergoing biopsy catheters were placed within viable tumour tissue (T). In all patients a second catheter was placed at least 20mm outside of the radiological bulk of the tumour in macroscopically normal brain around tumour (BAT). Microdialysates were analysed using the CMA 600 analyser, except for boron that was measured using an inductively coupled atomic emission spectrometer. The concentrations of boron varied considerably depending on the tissue sampled: in T samples the pharmacokinetic profile of BPA followed that of blood, while in BAT uptake was generally very low with a delay of up to 8 hours in relation to blood levels. No significant changes in glucose and its metabolites were noted during BNCT treatment. An increase in the concentration of glycerol was noted in T and PT 1-3 days after BNCT treatment while BAT levels were low and unchanged. Glutamate also showed high levels in PT compared to

In their second study the Swedish group evaluated the levels of glucose and its metabolites, glycerol and glutamate in patients with HGG undergoing conventional post-operative radiotherapy (Tabatabaei et al. 2008). Thirteen patients with HGG were recruited with one catheter placed within the tumour (T), and a second 10mm outside the contrast-enhancing area in the peritumour region (PT). Samples were obtained at least 20 hours before radiotherapy commenced, and then continued for at least 20 hours after the fifth radiotherapy session. Baseline levels of glucose were significantly lower, and the L/P ratio significantly higher, in dialysates from T compared with PT. Radiotherapy did not influence

Bergenheim's group subsequently extended their approach to utilise HWCO catheters enabling evaluation of macromolecules during conventional radiotherapy (Wibom et al.). Eleven patients with HGG were underwent stereotactic biopsy with insertion of two microdialysis catheters: one placed into the contrast enhancing tumour (T), and a second outside it in the peritumour region (PT). Reference samples were also collected subcutaneously from patients' abdomen. Microdialysates were analysed using gas chromatography – time-of-flight mass spectroscopy. Marked differences in metabolomic patterns were noted between T and PT, and between brain and abdominal microdialysates. In addition, dynamic changes occurred with radiotherapy in T and PT microdialysates.

A novel use of clinical microdialysis is to deliver chemotherapeutic agents through a process termed retrograde microdialysis. The technique offers a number of potential advantages. First, the precise placement of catheters allows chemotherapy to bypass the blood-brain barrier and be administered directly to the tumour bed. Second, equilibration occurs across a semi-permeable membrane ensuring the therapeutic concentration is maintained. Third, simultaneous measurement of metabolism allows direct assessment of a drug's effects. The therapeutic principle was first explored by Ungerstedt's group in Sweden who treated three patients with GBM by adding the oncotoxic non-physiological amino acid L-2, 4

BAT, although no obvious changes were observed over time.

glucose and its metabolites, or glycerol or glutamate.

**5. Retrograde microdialysis** 

with two solutions of different concentrations of rifampin at a rate of 3µl/m. The loss or gain of rifampin from the two solutions was determined and rifampin concentration then calculated using the no-net-flux method. Intra-operatively solid tissue samples were also taken from tumour tissue, peri-tumour tissue and unaffected brain around tumour tissue. The concentration of rifampin was greatest within solid tissue samples from tumour and peritumour, followed by BAT microdialysates, and then solid tissue samples from unaffected brain around tumour. The rifampin concentration in all compartments exceeded the minimum inhibitory concentration (MIC) for staphylococci and streptococci.

Blakeley et al used clinical microdialysis to investigate the pharmacokinetics of high dose methotrexate (12g/m2) (Blakeley et al. 2009). The group performed an in-vitro recovery experiment, which demonstrated a relative recovery of 43.6 ± 2.6%. Four patients with recurrent HGG were recruited and underwent biopsy or resection as clinically indicated. A LWCO microdialysis catheter was then placed into either the contrast-enhancing or nonenhancing residual tumour (T). Samples were analysed using liquid chromatography/mass spectroscopy. Methotrexate penetration in T was found to be variable with the highest concentrations measured within the contrast-enhancing regions. Nevertheless, the concentration of methotrexate in all regions exceeded the minimum concentration required for 50% cell kill against glioma cell lines in vitro.

Portnow et al used clinical microdialysis to investigate the pharamcokinetics of another chemotherapeutic drug temozolamide (TMZ) (Portnow et al. 2009). Contemporary postoperative management of patients with a HGG is with daily TMZ tablets and concurrent radiotherapy. Phase I studies of TMZ suggested that peak levels in blood occurred approximately an hour after ingestion and patients are therefore typically instructed to take their tablets an hour prior to radiotherapy to potentiate their oncotoxic effects (Dhodapkar et al. 1997). The group first performed an in-vitro recovery experiment, which demonstrated a relative recovery of 87 ± 5.5%. Portnow et al then recruited 9 patients of which 6 patients had HGG, and 3 had non-small cell lung cancer. All patients underwent resection and a single LWCO microdialysis catheter was placed within 5mm of the tumour resection margin (T). Post-operatively one patient refused TMZ, and in another the microdialysis catheter was occluded. In the remaining 7 patients microdialysates were analysed using HPLC to determine the concentration of TMZ. Concentrations of temozolamide in the brain measured in their study were consistent with previous studies but it was noted that the mean time to reach peak level in the brain was 2.0 ± 0.8 hour. The clinical corollary of their findings is that current chemoradiation regimens may be improved by advising patients to take their tablets 2 hours before radiotherapy sessions.

#### **4.2 Radiotherapy**

A series of studies by a group in Sweden have used microdialysis to monitor patients undergoing radiotherapy. The focus of their first study was Boron Neutron Capture Therapy (BNCT), an experimental technique in which patients are injected with boron, which preferentially binds to tumour cells, and then treated with neutron beam radiotherapy generating oncotoxic alpha particles and Lithium ions. The technique is theoretically attractive because of the short path length of alpha particles (approximately one cell diameter) compared to conventional gamma radiation. Bergenheim et al used clinical microdialysis to determine the pharmacokinetics of boronophenylalanine (BPA) with a view to optimising the timing of radiation (Bergenheim et al. 2005). An in-vitro

with two solutions of different concentrations of rifampin at a rate of 3µl/m. The loss or gain of rifampin from the two solutions was determined and rifampin concentration then calculated using the no-net-flux method. Intra-operatively solid tissue samples were also taken from tumour tissue, peri-tumour tissue and unaffected brain around tumour tissue. The concentration of rifampin was greatest within solid tissue samples from tumour and peritumour, followed by BAT microdialysates, and then solid tissue samples from unaffected brain around tumour. The rifampin concentration in all compartments exceeded

Blakeley et al used clinical microdialysis to investigate the pharmacokinetics of high dose methotrexate (12g/m2) (Blakeley et al. 2009). The group performed an in-vitro recovery experiment, which demonstrated a relative recovery of 43.6 ± 2.6%. Four patients with recurrent HGG were recruited and underwent biopsy or resection as clinically indicated. A LWCO microdialysis catheter was then placed into either the contrast-enhancing or nonenhancing residual tumour (T). Samples were analysed using liquid chromatography/mass spectroscopy. Methotrexate penetration in T was found to be variable with the highest concentrations measured within the contrast-enhancing regions. Nevertheless, the concentration of methotrexate in all regions exceeded the minimum concentration required

Portnow et al used clinical microdialysis to investigate the pharamcokinetics of another chemotherapeutic drug temozolamide (TMZ) (Portnow et al. 2009). Contemporary postoperative management of patients with a HGG is with daily TMZ tablets and concurrent radiotherapy. Phase I studies of TMZ suggested that peak levels in blood occurred approximately an hour after ingestion and patients are therefore typically instructed to take their tablets an hour prior to radiotherapy to potentiate their oncotoxic effects (Dhodapkar et al. 1997). The group first performed an in-vitro recovery experiment, which demonstrated a relative recovery of 87 ± 5.5%. Portnow et al then recruited 9 patients of which 6 patients had HGG, and 3 had non-small cell lung cancer. All patients underwent resection and a single LWCO microdialysis catheter was placed within 5mm of the tumour resection margin (T). Post-operatively one patient refused TMZ, and in another the microdialysis catheter was occluded. In the remaining 7 patients microdialysates were analysed using HPLC to determine the concentration of TMZ. Concentrations of temozolamide in the brain measured in their study were consistent with previous studies but it was noted that the mean time to reach peak level in the brain was 2.0 ± 0.8 hour. The clinical corollary of their findings is that current chemoradiation regimens may be improved by advising patients to

A series of studies by a group in Sweden have used microdialysis to monitor patients undergoing radiotherapy. The focus of their first study was Boron Neutron Capture Therapy (BNCT), an experimental technique in which patients are injected with boron, which preferentially binds to tumour cells, and then treated with neutron beam radiotherapy generating oncotoxic alpha particles and Lithium ions. The technique is theoretically attractive because of the short path length of alpha particles (approximately one cell diameter) compared to conventional gamma radiation. Bergenheim et al used clinical microdialysis to determine the pharmacokinetics of boronophenylalanine (BPA) with a view to optimising the timing of radiation (Bergenheim et al. 2005). An in-vitro

the minimum inhibitory concentration (MIC) for staphylococci and streptococci.

for 50% cell kill against glioma cell lines in vitro.

take their tablets 2 hours before radiotherapy sessions.

**4.2 Radiotherapy** 

experiment determined a mean BPA recovery of 66.8 ± 8.8%. The group also monitored glucose and its metabolites, glutamate and glycerol throughout the procedure. Four patients with WHO grade IV glioblastoma multiforme (GBM) were recruited. One patient underwent gross total resection, one a subtotal resection, and two stereotactic biopsies. In the patients that underwent resection a microdialysis catheter was placed within 5mm of the resection margin (T), and in the patients undergoing biopsy catheters were placed within viable tumour tissue (T). In all patients a second catheter was placed at least 20mm outside of the radiological bulk of the tumour in macroscopically normal brain around tumour (BAT). Microdialysates were analysed using the CMA 600 analyser, except for boron that was measured using an inductively coupled atomic emission spectrometer. The concentrations of boron varied considerably depending on the tissue sampled: in T samples the pharmacokinetic profile of BPA followed that of blood, while in BAT uptake was generally very low with a delay of up to 8 hours in relation to blood levels. No significant changes in glucose and its metabolites were noted during BNCT treatment. An increase in the concentration of glycerol was noted in T and PT 1-3 days after BNCT treatment while BAT levels were low and unchanged. Glutamate also showed high levels in PT compared to BAT, although no obvious changes were observed over time.

In their second study the Swedish group evaluated the levels of glucose and its metabolites, glycerol and glutamate in patients with HGG undergoing conventional post-operative radiotherapy (Tabatabaei et al. 2008). Thirteen patients with HGG were recruited with one catheter placed within the tumour (T), and a second 10mm outside the contrast-enhancing area in the peritumour region (PT). Samples were obtained at least 20 hours before radiotherapy commenced, and then continued for at least 20 hours after the fifth radiotherapy session. Baseline levels of glucose were significantly lower, and the L/P ratio significantly higher, in dialysates from T compared with PT. Radiotherapy did not influence glucose and its metabolites, or glycerol or glutamate.

Bergenheim's group subsequently extended their approach to utilise HWCO catheters enabling evaluation of macromolecules during conventional radiotherapy (Wibom et al.). Eleven patients with HGG were underwent stereotactic biopsy with insertion of two microdialysis catheters: one placed into the contrast enhancing tumour (T), and a second outside it in the peritumour region (PT). Reference samples were also collected subcutaneously from patients' abdomen. Microdialysates were analysed using gas chromatography – time-of-flight mass spectroscopy. Marked differences in metabolomic patterns were noted between T and PT, and between brain and abdominal microdialysates. In addition, dynamic changes occurred with radiotherapy in T and PT microdialysates.

### **5. Retrograde microdialysis**

A novel use of clinical microdialysis is to deliver chemotherapeutic agents through a process termed retrograde microdialysis. The technique offers a number of potential advantages. First, the precise placement of catheters allows chemotherapy to bypass the blood-brain barrier and be administered directly to the tumour bed. Second, equilibration occurs across a semi-permeable membrane ensuring the therapeutic concentration is maintained. Third, simultaneous measurement of metabolism allows direct assessment of a drug's effects. The therapeutic principle was first explored by Ungerstedt's group in Sweden who treated three patients with GBM by adding the oncotoxic non-physiological amino acid L-2, 4

Clinical Microdialysis in Glioma 157

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daminobutyric acid (DAB) to perfusate (Ronquist et al. 1992). This was extended by Bergenheim et al who recruited 10 patients with GBM that underwent stereotactic biopsy with the insertion of two microdialysis catheters: one in the contrast enhancing tumour (T), and the second 10mm outside the contrast enhancing region in the peritumour region (PT) (Bergenheim et al. 2006). Catheters inserted into T were LWCO with 30mm membranes, and were perfused with 80 or 120mmol/l DAB at a rate of 2µl/m. Samples were analysed for metabolites using a CMA 600 analyser, and for amino acids using HPLC. During treatment with DAB a significant increase in a number of amino acids including glutamate was observed suggesting cellular toxicity. PT samples were unaffected suggesting treatment effects was localised to the tumour compartment. Although the sample size was too small to determine whether there was an effect on clinical outcome, the study nevertheless provides evidence to support the feasibility of the technique.
