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Methotrexate Accumulates to Similar Levels in Animals Transplanted with Normal versus Drug-resistant Transgenic Marrow¹

Gene Therapy Program, Institute of Human Genetics [L. R. B., M. D. D., R. S. M.], Department of Genetics, Cell Biology and Development [L. R. B., R. S. M.], and Department of Pharmaceutics, College of Pharmacy [D. B-G., C. L. Z.], University of Minnesota, Minneapolis, Minnesota 55455

Gene transfer and expression of methotrexate (MTX)-resistant variants of dihydrofolate reductase (DHFR) in normal hematopoietic cells is a potential strategy to permit administration of larger doses of MTX by alleviating drug toxicity in normal cells and tissues that are drug sensitive. We have previously demonstrated that transplantation of marrow from transgenic mice expressing drug-resistant DHFRs conferred upon normal recipient animals resistance to MTX at levels that are usually toxic for hematopoietic and gastrointestinal (GI) tissues. One explanation for the observed protection from GI toxicity by drug-resistant marrow is that MTX could be cleared more rapidly in animals maintaining a more healthy hematopoietic system. To evaluate this possibility, we carried out MTX pharmacokinetic studies in mice that received transplanted transgenic marrow expressing either of two different DHFR variants, administering increasing doses of MTX up to 4 mg/kg/day. Animals received i.p. injection precisely every 24 h. Every 4 days, three animals from each group were sacrificed, and their plasma and intestines were assayed for MTX. Animals transplanted with transgenic Arg-22 DHFR drug-resistant marrow maintained hematocrit levels that were about 4-fold higher at 3 weeks after transplant than those of untreated animals or animals that received normal marrow cells. Animals that received normal marrow did not survive beyond 25 days and did not accumulate higher levels of MTX than animals that received a transgenic marrow transplant. Untreated animals exhibited a higher rate of survival (36 days) but again did not accumulate higher levels of MTX than the transgenic marrow recipients. When the experiment was repeated using transgenic Tyr-22 DHFR marrow, the levels of MTX in the plasma or GI tissues did not differ significantly between groups. Intestinal concentrations of MTX in both experiments were about 4–5-fold higher than those in the plasma. These results indicate that protection from MTX toxicity conferred by expression of drug-resistant DHFR activity in the marrow is not the result of a higher rate of MTX clearance from the circulation in comparison with control animals but a true resistance of hematopoietic and GI tissues to MTX. The maintenance of antifolate levels in animals protected from MTX toxicity implies that this procedure should not compromise the antitumor efficacy of MTX.