COVALENT COUPLING OF METHOTREXATE TO DEXTRAN ENHANCES THE PENETRATIONOF CYTOTOXICITY INTO A TISSUE-LIKE MATRIX

For antitumor agents introduced directly into the intracranial space, the extent of penetration into tissue, and hence the effectiveness of therapy, depends on the rate of drug elimination from the tissue. To t est the hypothesis that slowly eliminated agents would penetrate furth er through tissues, methotrexate (MTX)-dextran conjugates were produce d by covalently linking MTX to dextran through a short-lived ester bon d (MTX-ester-dextran; t1/2 approximately 3 days in buffered saline) an d a longer-lived amide bond (MTX-amide-dextran; t1/2 > 20 days in buff ered saline). The ability of these agents to kill cells and to penetra te through tissue was evaluated using: (a) human brain tumor (H80) cel ls in a standard format: (b) H80 cells in a novel three-dimensional fo rmat that mimics many characteristics of intracranial tumors; and (c) 9L gliosarcoma in the rat brain. Penetration into three-dimensional ti ssue-like matrices was performed by suspending H80 cells in agarose ge ls within a hollow fiber that was permeable to MTX but not dextran and injecting MTX or MTX-dextran conjugates into one end of the fiber. Th e cytotoxicity of MTX-ester-dextran and MTX-amide-dextran against H80 was equivalent to unmodified MTX (50% inhibitory concentration, approx imately 0.01 mug/ml). When released from a biodegradable polyanhydride polymer matrix, MTX and MTX-dextran conjugates retained their ability to inhibit dihydrofolate reductase activity. When MTX or MTX-dextran was diffused into the three-dimensional tumor cell matrix for 10 days, cytotoxic activity penetrated >2 cm for MTX-amide-dextran and approxi mately 1 cm for MTX or MTX-ester-dextran; this enhanced penetration co rrelated with the stability of the MTX-dextran linkage. Intracranial p olymeric delivery of MTX or MTX-amide-dextran to rats with intracrania l 9L gliosarcoma produced modest but significant increases in survival ; conjugation of MTX to dextrin appeared to shift the dose-response cu rve to a lower dosage.