1. Delayed Neurotoxicity The cumulative risk at 5 years to develop overt dementia is 24-30% (1,2). For patients aged >60 year the risk of dementia at 7 years is 58% (2). Multivariate analysis shows that only radiotherapy is a significant factor related to neurotoxicity (1). By 4 years fatal neurotoxicity rate is 10% (3). (1) Omuro A.M. et al: Arch Neurol 62:1595-1600, 2005 (2) O’Brien P.C. et al: Int J Rad Oncol Biol Phys 64:408-16, 2006 (3) Fisher B et al: J Neuro-Oncol 74:201-205, 2005

2. Delayed Neurotoxicity By 4 years fatal neurotoxicity rate is 10% Modification of the RT schedule to a 25% reduction in biologically effective tumor dose (36Gy/30 fractions/3 wks) – delayed but did not eliminate fatal neurotoxicity Reduction of RT dose did not compromise treatment outcome Fisher B et al: J Neuro-Oncol 74:201-205, 2005

3. Delayed Neurotoxicity O’Brien P.C. et al: Int J Rad Oncol Biol Phys 64:408-13, 2006

4. Fractional Delivery of Chemotherapeutic Agents Across the BBB and BTB Local Exposure Fraction MTX 0.0170.18 5-FU 0.110.25 AZQ 0.700.72 BCNU 0.540.55 Drug Normal Brain Increased Permeability = 0.1 ml/g/min

5. Methods to Increase Drug Delivery Increase delivery of systemic drug administration (intravascular drugs) Increase drug delivery by local administration (intra-CSF; intra-parenchymal-CED)

6.  Manipulating the drug (chemical modifications, prodrugs)  Increasing the fraction of the drug reaching the tumor (High-dose chemotherapy, intra-arterial administration)  Manipulating the capillary permeability (osmotic BBBD, chemical modification of BBB/BTB, receptor mediated transport) Increase Delivery of Intravascular Drugs

7. Manipulating the drug (chemical modifications, prodrugs)

8. Manipulating the drug (chemical modifications, prodrugs) Increase the fraction of the drug reaching the tumor (High-dose chemotherapy, intra-arterial administration) Manipulating the capillary permeability (osmotic BBBD, chemical modification of BBB/BTB, receptor mediated transport) Increase delivery of intravascular drugs

9. Increase the Fraction of the Drug Reaching the Tumor: HD-Chemotherapy Increase plasma concentration with systemic rescue maneuvers  High-dose MTX + folinic acid rescue  High-dose Ara-C + granulocytic growth factor  High dose chemotherapy with stem cell support Rescue maneuvers reduce systemic toxicity and make the treatment relatively safe. Still, systemic toxicity is the major limiting factor.

10. Several studies tried to use intensive chemotherapy as the sole treatment for PCNSL but their limitations proved to be either:  A low response rate (25% CR) (single agent HD-MTX) (1)  A short duration of response (2)  A high rate of treatment-related death (9-10%) (3). High Dose Chemotherapy in PCNSL (1) Herrlinger U. et al: Ann Neurol 51:247-252, 2002 (2) Abrey L..E. et al: J Clin Oncol 21: 4151-4156, 2003 (3) Pels H. et al: J Clin Oncol 21: 4489-4495, 2003

11. % of treatment cycles Author I.V. MTX dose with CSF MTX level (gr/m2) > 1 mmol/L Thyss-1987 0.5 0% Thyss-1987 2.5 44% Millot -1994 5.0 66% Borsi -1987 6.0 100% Pharmacokinetics of HD-MTX Human Studies

12. CSF MTX concentrations: marked interindividual variability. Cytotoxic concentrations are obtained with marked increment of the total dose (to 6-8 gr/m2). The mean AUC ratio (CSF/serum) MTX: 1.52 - 3.0% (systemic dose: 5 - 8 gr/m2 ) reflecting the poor diffusion of MTX into the CNS.

13. MTX Pharmacokinetics Human studies evaluated CSF MTX. Peak CSF MTX concentrations are observed between 4-6 hrs after MTX infusion while serum levels are rapidly declining. Do CSF MTX levels reflect parenchymal drug penetration? CSF peak levels may reflect the sink effect or the washout of the drug from the brain and tumor extracellular space.

14. Methotrexate Pharmacokinetics in Dog After BBBD Neuwelt et al, Neurosurgery 7:36-43,1980 CSF Brain

15. Pharmacokinetics of MTX in the ECF of Brain Tumor Plasma total MTX Plasma free MTX ECF MTX i.v. MTX infusion Dukic et al, Pharmaceutical Res 16:1219-25,1999 AUC ECF /AUC Plasma 1.02 + 0.75 % (0.36 -2.37) AUC ECF /AUC Plasma 1.02 + 0.75 % (0.36 -2.37)

16. MTX Penetration in Brain Tumor AUC ECF /AUC Plasma i.v. bolus: 4.15 + 2.20 % i.v. infusion: 1.39 + 0.79 % AUC ECF /AUC Plasma i.v. bolus: 4.15 + 2.20 % i.v. infusion: 1.39 + 0.79 % Dukic et al, Europ J Cancer 36:1578-84,2000 Plasma ECF Bolus

17. n=13 n=16 PCNSL and HD-MTX: Rapid (3 hrs) vs. Regular (6 hrs) Infusion of MTX Rapid Regular CSF MTX ConcentrationTumor VolumeRelapse-Free Survival Higara et al, J Neurosurgery 91:221-30,1999 P<0.001 Rapid Regular P<0.001