Removal Of Methotrexate Research Articles

Effects of hemoperfusion plus high-flux hemodialysis in a patient with methotrexate toxicity

Acute methotrexate toxicity resulting from methotrexate-induced renal failue is a medical emergency requiring extracorporeal removal of methotrexate. The optimum method of methotrexate removal has not yet been established. We report a case of osteosarcoma with lung metastasis that was treated with high-dose methotrexate as adjuvant chemotherapy. Although no problems occurred after the first 5 courses of methotrexate, methotrexate-induced renal failure and methotrexate toxicity appeared after the sixth course. The patient was treated, either with hemoperfusion plus high-flux hemodialysis, or hemoperfusion alone, and pre- and post-treatment serum methotrexate concentrations were monitored. The reduction in methotrexate by hemoperfusion alone for 2 hours was 54%, compared to a mean reduction of 59% by hemoperfusion combined with high-flux hemodialysis, for 3 hours. Rebound increases in methotrexate levels were small (less than 1 μmol/L) with either method. The combination of hemoperfusion and hemodialysis resulted in good control of volume status, as well as improvement in serum chemistry values.

A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.

Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.

Comparison of Hemodialysis versus Hemoperfusion in the Clearance of High‐Dose Methotrexate in Pigs

To evaluate the different capabilities of hemo-perfusion (HP) and hemodialysis (HD) treatment in resolving the side effects during high-dose methotrexate (MTX) therapy, an experimental comparative study was done. Under general anesthesia, 12 female Large-White pigs, 35 +/- 5 kg body weight, underwent a bilateral renal vessel ligature to create an acute renal failure and avoid the physiologic elimination of MTX. An MTX i.v. infusion was performed using a dose of 12.5 g/m2 and for a specific time according to its pharmacokinetics. The animals were divided into 2 groups of 6 animals each and connected to the extracorporeal circuit; the first group was submitted to a 1-h HP with an anion exchange resin (Dow 1X-2, Dow Chemical) and the second group to 1-h HD with capillary polysulfone fibers (F6, Fresenius). Blood samples were taken at established intervals for hematological and biochemical evaluations. The results demonstrate a higher capability of HP treatment (P < 0.001) related to HD in MTX removal.

Enhanced expression of dihydrofolate reductase by bovine kidney epithelial cells results in altered cell morphology, IGF-I responsiveness, and IGF binding protein-3 expression.

The kidney epithelial cell line (MDBK) secretes primarily insulin-like growth factor binding protein (IGFBP)-2 under basal conditions, but exposure to forskolin decreases the synthesis of and induces IGFBP-3. Since IGFBP-3 has been shown to both potentiate and inhibit insulin-like growth factor (IGF) bioactivity, MDBK cells were transfected with an expression vector containing bovine IGFBP-3 cDNA and the dihydrofolate reductase (DHFR) gene as a selectable marker, with the goal of obtaining an epithelial cell line which constitutively secreted IGFBP-3. Stable clones which secreted greater than 100 ng/ml of IGFBP-3 were obtained and designated MDBKpMONBP-3. Northern blotting indicated that endogenous IGFBP-3 mRNA, which was undetectable in wild-type (WT) MDBK cells, was expressed in MDBKpMONBP-3 cells while the IGFBP-3 transgene did not appear to be expressed. DHFR mRNA transcripts were also expressed by MDBKp-MONBP-3 cells, whereas these transcripts were not detected in WT MDBK cells, suggesting that gene amplification of DHFR may have allowed cells to survive in methotrexate (MTX) without taking up the expression vector. In addition to the altered pattern of IGFBP-3 secretion, a marked alteration in cell morphology was observed. MDBKpMONBP-3 cells grew in distinct islands and exhibited dome formation (a characteristic of differentiated epithelial cells) whereas the WT cells did not. The alterations in morphology and IGFBP-3 expression were irreversible, since MDBKpMONBP-3 cells failed to revert to the WT phenotype upon removal of MTX and dialyzed serum. Since vectorial secretion of proteins is often associated with epithelial cell differentiation, cells were plated on tissue culture inserts which allowed conditioned media (CM) to be collected from both the apical and basal surfaces of confluent monolayers. Release of IGFBP-2 was approximately equal from apical and basal surfaces in WT MDBK cells. In contrast, release of both IGFBP-2 and IGFBP-3 was greater (3.1-fold and 3.5-fold, respectively) from basal as compared to apical surfaces of the MDBKpMONBP-3 cells. To determine if cells which were secreting IGFBP-3 had altered growth responses to IGF-I, cells were grown in serum-free media in the presence of IGF-I (0 to 100 ng/ml). Treatment of MDBKpMONBP-3 cells with 100 ng/ml of IGF-I increased cell number 138 +/- 37% above serum-free controls compared to 73 +/- 10% in WT MDBK cells.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro and ex vivo evaluation of methotrexate removal by different sorbents haemoperfusion.

The administration of high doses of Methotrexate (HDMTX) seems to be very effective in neoplastic treatment. Furthermore high doses of the drug could overcome the Methotrexate (MTX) resistance that it is possible to observe in some kind of cancers. To avoid several serious problems in the therapeutical application of HDMTX such as nephrotoxicity, hepatotoxicity and the difficulty to assess the right therapeutic range of the specific antagonist Leucovorin, a Haemoperfusion (HP) treatment could be very usefulL. In this study 3 anion exchange resins with different active group (trimethylammonium, dimethylethanolammonium and dimethylammina) and a spherical petroleum based charcoal were tested both in vitro and ex vivo to evaluate their capability to remove MTX. These results show a good strength and biocompatibility of the 4 sorbents and demonstrate the active group trimethylammonium resin as the most effective one.

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

The effect of methionine on methotrexate metabolism in rat hepatocytes in monolayer culture

The effect of methyl donors on the metabolism of methotrexate has been investigated in rat hepatocytes in monolayer culture. Pulse exposure to low concentrations of methotrexate (1 μM, 3 h) in the absence of methionine results in the facile formation of the di- to pentaglutamates with the di- and triglutamate predominating. Further incubation after the removal of methotrexate (MTX) results in a shift to the tetra- and pentaglutamate at the expense of the shorter chain length derivatives. The same measurement in the presence of 1 mM methionine causes approx. an 80% inhibition in the formation of polyglutamates. This effect can be partially achieved when methionine is replaced by choline or betaine. No alteration in the formation of 7-hydroxymethotrexate could be detected by similar changes in methionine concentrations in the medium. The activity of the enzymes which synthesize and degrade methotrexate polyglutamates, folypolyglutamate synthetase and γ-glutamyl hydrolase, respectively, were the same in extracts of cells grown in the absence and in the presence of 1 mM methionine. Incubation of the hepatocytes with methionine causes a significant increase in 5,6,7,8-tetrahydrofolate (H 4folate), 5,10-methylenehydrofolate and 10-formyltetrahydrofolate and a decrease in 5-methyltetrahydrofolate. These results suggest that the inhibition of glutamylation of methotrexate could be due in part to an elevation in reduced folates which can more effectively compete with methotrexate as a substrate for folypolyglutamate synthetase. Inhibition in methotrexate glutamylation by methionine, betaine and choline in hepatocytes may contribute to the alleviation of hepatic toxicity by methyl donors.

Removal of methotrexate by hemodiafiltration

Removal of methotrexate by hemodiafiltration

Ribosomal RNA gene amplification: A selective advantage in tissue culture

A CCRF-CEM (human T-cell leukemia) line, initially made resistant to methotrexate by a four-step increase in drug concentration, revealed a large marker of one chromosome #14 in all cells after a 6-month period of culture in the absence of drug. The marker is the result of a triple duplication of both the satellite and stalk. In situ hybridization with 3H-cRNA and NOR banding proved that ribosomal DNA had been amplified and was actively transcribed. Flow cytometric analysis showed a significant increase of RNA versus DNA in G 1 cells compared with those of the original drug-resistant parent. The abnormal chromosome appeared after the removal of methotrexate from culture and may be related to the faster doubling time of this line compared with the parent line.

Functional resistance of inflammatory macrophages to methotrexate in vitro.

The purpose of this study was to evaluate the effects of high and low therapeutic doses of methotrexate (MTX) on macrophage metabolism and function in vitro. Monolayers of elicited rat peritoneal macrophages (PM) were exposed to a wide range of MTX concentrations (10(-8) M-10(-3) M) for 24 or 48 hr and macrophage RNA and protein metabolism were evaluated by the incorporation of [3H]5-uridine and [14C]1-leucine, respectively, into trichloroacetic acid (TCA)-precipitable material. Macrophage functional activity was examined by measuring the uptake of [14C]Pseudomonas aeruginosa to assess phagocytosis and the release of 51Cr from antibody-coated [51Cr]sheep red blood cells (SRBC) to assess antibody-dependent cell-mediated cytotoxicity. Following a 24-hr incubation with 10(-3) M MTX, incorporation of [3H]5-uridine into PM monolayers was enhanced 79% when compared to control monolayers (p less than 0.005). Washout studies revealed that the stimulation of uridine incorporation was no longer observed by 24 hr following the removal of MTX from the culture medium. Incubation with 10(-3) M MTX for 48 hr returned uridine incorporation to control levels, although leucine incorporation into protein was reduced by 22% (p less than 0.01). The depression in leucine incorporation in the presence of 10(-3) M MTX was not reversed after the removal of MTX from the culture medium. Uptake of [14C]P. aeruginosa was not altered following a 24- or 48-hr incubation with either 10(-7) M or 10(-3) M MTX. Similarly, [51Cr]SRBC cytolysis was not affected by a 2-hr preincubation with and continuous exposure to between 10(-8) M and 10(-3) M MTX. These results demonstrate that incubation of inflammatory macrophages with clinically high doses of MTX can alter macrophage RNA and protein metabolism without producing demonstrable changes in macrophage functional activity.

Methotrexate permits a limited number of cell doublings and affects mitochondrial substructure inTetrahymena

Methotrexate (MTX), a folic acid analogue, is used in cancer chemotherapy in low or high doses. Addition of MTX to proliferatingTetrahymena cultures revealed that, irrespective of the concentration of MTX (1–50 mM), the cells doubled once during a 6-hour exposure and twice during a 24-hour exposure; the normal generation time is 3 hours. MTX had a dose-dependent effect on the maximal number of cell doublings reached after 72 hours, thus 5 1/2 and 3 1/2 doublings were found in 1 and 10 mM MTX, respectively. Massive cell death was observed after a 4-day exposure. At all concentrations of MTX, the rate of endocytosis was unaffected initially but decreased to 60% of the control value after 24 hours. Conspicuously, small refractive, or electron dense, granules accumulated in MTX-treated cells; these granules are presumably lysosomes accumulating MTX for defecation. The mitochondrial substructure became altered in MTX-treated cells; after a 24-hour exposure to MTX the mitochondria were almost depleted of tubules. This reduction of the number of mitochondrial tubules occurred gradually, apparently correlated with division of the organelles. Recovery was also studied of cells exposed for 1 or 3 hours to 10 mM MTX. During the first 24 hours after removal of MTX, the cells were affected in a manner similar to that described for cells exposed continuously to MTX, also with respect to the altered substructure of mitochondria; however, the recovering cells resumed the normal rate of cell proliferation after 4 cell doublings. These longlasting effects, of even a short exposure ofTetrahymena to MTX, may in part explain the cause of the severe side effects accompanying MTX chemotherapy.

Anisotropic membranes with carboxypeptidase G1.

Anisotropic polysulfone membranes were prepared with carboxypeptidase G1 embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.

The kinetics of methotrexate polyglutamate formation and efflux in a human breast cancer cell line (MDA.MB.436): The effect of insulin

The rate and extent of the formation of methotrexate poly-γ-glutamates has been studied in a human breast cancer cell line (MDA.MB.436). Cells were exposed to medium containing 10 −7 M radiolabelled methotrexate (MTX) for various periods. Cell extracts were subjected to gel filtration on Bio-Gel P2 and radioactivity in each fraction determined. This has allowed quantitation of the relative amounts of MTX and its polyglutamate derivatives. It has been proposed that MTX polyglutamates may act as storage forms of the drug which could result in prolonged cytotoxicity. This possibility was investigated by determining the ability of the cell line to retain MTX and its olyglutamate derivatives for periods of up to 48 hr after removal of MTX from the incubation medium. Our results show that the lower mol. wt polyglutamates (< three extra glutamic acid residues) rapidly efflux from the cell, while the higher mol. wt species (> three glutamic acid residues) are extensively retained, having an efflux half-life of approximately 30 hr. It has been shown that insulin may potentiate the cytotoxicity of MTX to breast cancer cells in vitro. Our results indicate that in the MDA.MB.436 cell line the total intracellular drug at the steady state is unaffected by insulin (10 −6 M). However insulin does result in a 30% increase in the contribution of the higher mol. wt polyglutamates to the total intracellular drug. Our data therefore suggest that the ability of insulin to potentiate the cytotoxic effects of MTX may be related to the hormone's ability to modulate the synthesis of MTX polyglutamates.

Will hemoperfusion be useful for cancer chemotherapeutic drug removal?

One of the major problems in clinical cancer chemotherapy is the inability to safely administer full therapeutic doses of specific drugs in the face of dysfunction of an organ system controlling that drug's metabolism and excretion. Should efficient drug removal from blood be possible following full therapeutic doses and after tumor exposure, then theoretically, even in the presence of organ dysfunction, anticancer drug toxicity may be reduced or avoided. Preliminary experiments in our laboratory have shown that adriamycin may be efficiently removed by activated charcoal from aqueous and protein solutions and blood in vivo, and that daunorubicin is removed in vitro to the same extent. However, although methotrexate is removed efficiently in vitro and extracted 50% in vivo by charcoal hemoperfusion, its overall pharmacokinetics do not appear to be altered in comparison with the alteration in pharmacokinetics of adriamycin achieved with charcoal hemoperfusion. Computer modeling has suggested that efficient adriamycin removal is achievable, and that clinical studies are warranted. For methotrexate removal, however, previous clinical studies and our own data suggest that charcoal hemoperfusion has little utility unless a highly specific sorbent for methotrexate removal can be developed.