Methotrexate accumulates to similar levels in animals transplanted with normal versus drug-resistant transgenic marrow

Gene transfer and expression of methotrexate (MTX)-resistant variants of di hydrofolate reductase (DHFR) in normal hematopoietic cells is a potential s trategy to permit administration of larger doses of MTX by alleviating drug toxicity in normal cells and tissues that are drug sensitive. We have prev iously demonstrated that transplantation of marrow from transgenic mice exp ress ing drug-resistant DHFRs conferred upon normal recipient animals resis tance to MTX at levels that are usually toxic for hematopoietic and gastroi ntestinal (GT) tissues. One explanation for the observed protection from GI toxicity by drug-resistant marrow is that MTX could be cleared more rapidl y in animals maintaining a more healthy hematopoietic system. To evaluate t his possibility, we carried out MTX pharmacokinetic studies in mice that re ceived transplanted transgenic marrow expressing either of two different DH FR variants, administering increasing doses of MTX up to 4 mg/kg/day, Anima ls received i.p. injection precisely every 24 h, Every 4 days, three animal s from each group were sacrificed, and their plasma and intestines were ass ayed for MTX. Animals transplanted with transgenic Arg-22 DHFR drug-resista nt marrow maintained hematocrit levels that were about 4-fold higher at 3 w eeks after transplant than those of untreated animals or animals that recei ved normal marrow cells. Animals that received normal marrow did not surviv e beyond 25 days and did not accumulate higher levels of MTX than animals t hat received a transgenic marrow transplant. Untreated animals exhibited a higher rate of survival (36 days) but again did not accumulate higher level s of MTX than the transgenic marrow recipients. When the experiment was rep eated using transgenic Tyr-22 DHFR marrow, the levels of MTX in the plasma or GI tissues did not differ significantly between groups. Intestinal conce ntrations of MTX in both experiments were about 4-5-fold higher than those in the plasma. These results indicate that protection from MTX toxicity con ferred by expression of drug-resistant DHFR activity in the marrow is not t he result of a higher rate of MTX clearance from the circulation in compari son with control animals but a true resistance of hematopoietic and GI tiss ues to MTX. The maintenance of antifolate levels in animals protected from MTX toxicity implies that this procedure should not compromise the antitumo r efficacy of MTX.