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PubMed1986

Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.

Aminobutyrates↗

PubMed1982

A comparison of the inhibition of growth of methotrexate-resistant and -sensitive leukemia cells in culture by triazines. Evidence for a new mechanism of cell resistance to methotrexate.

Forty-five 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-X-phenyl)-s-triazine inhibitors of dihydrofolate reductase (DHFR) and methotrexate (MTX) were tested on L5178Y/R murine tumor cell culture. The concentration of inhibitor causing a 50% decrease in growth rate was determined, and from these results a quantitative structure-activity relationship (QSAR) was developed. This QSAR is compared with QSAR for the same inhibitors acting on isolated DHFR and on L5178Y cell culture sensitive to MTX. The results show that very potent triazine inhibitors of resistant tumor cell growth can easily be made by making the triazines strongly hydrophobic. The optimum pi value for inhibition of MTX-sensitive cell culture is 0.8, while pi 0 for the resistant cell culture is about 6.0. The QSAR for MTX-sensitive and -resistant tumor cell culture inhibition is compared with the corresponding QSAR for Lactobacillus casei cells. Both the mammalian and bacterial cells appear to protect themselves from the highly hydrophilic MTX by erecting lipophilic barriers.

Animals↗

PubMed1982

Methotrexate analogues. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductase.

N alpha-(4-Amino-4-deoxy-N10-methylpteroyl)-N epsilon-(iodoacetyl)-L-lysine (1) was synthesized as a potential active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR). In an ultraviolet spectrophotometric assay of dihydrofolate reduction of Lactobacillus casei DHFR, 1 and methotrexate (MTX, 4-amino-4-deoxy-N10-methylpteroyl-L-glutamic acid) had ID50 values of 4.5 and 6.2 nM. The corresponding ID50 values in a competitive radioligand binding assay against [3H]MTX were 31 and 16 nM. Thus, as reversible inhibitors of this enzyme over a short exposure time, 1 and MTX had comparable activity. On the other hand, when L. casei DHFR was incubated for up to 6 h with 0.1 or 1.0 microM 1, a progressive decrease in the ability of [3H]MTX to subsequently displace the drug was observed. When MTX itself was used at the same concentrations, the extent of displacement of [3H]MTX did not decrease with time. These results were consistent with rapid reversible binding of 1 to the enzyme, followed more slowly by covalent bond formation near the active site. The pH profile for this effect followed a curve with a sigmoidal shape. The apparent inflection point near pH 7.2 was consistent with alkylation of a histidine residue.

Binding Sites↗

PubMed1983

Methotrexate analogues. 18. Enhancement of the antitumor effect of methotrexate and 3',5'-dichloromethotrexate by the use of lipid-soluble diesters.

Lipophilic methotrexate (MTX) and 3',5'-dichloromethotrexate (DCM) diesters were prepared by HCl-catalyzed esterification or by neutral esterification using cesium carbonate and an alkyl or aralkyl halide in Me2SO. The products were tested for in vivo antitumor activity against L1210 leukemia in mice to test whether all MTX and DCM diesters are therapeutically equivalent in this species. Contrary to what has been found with simple primary dialkyl esters, ortho-substituted dibenzyl esters of MTX produce longer survival on a q3dX3 schedule than does MTX itself and show a dose-sparing effect comparable to that of MTX at shorter treatment intervals. Thus, MTX bis(6-chloropiperonyl) ester at an MTX-equivalent dose of 5.5 mg/kg gave a +88% increase in median life span (ILS), whereas for MTX a +88% ILS required 30 mg/kg. When the MTX-equivalent dose of MTX bis(6-chloropiperonyl) ester was increased to 40 mg/kg, a +167% ILS was observed, as compared with a +100% ILS with 60 mg/kg of the parent drug. High activity (greater than 100% ILS) was likewise shown by the bis(2,5-dimethylbenzyl), bis(2,6-dichlorobenzyl), and di-3-picolyl esters of MTX and by the bis(1-methylbutyl) ester of DCM. The results of this study indicate that MTX and DCM esters are not therapeutically equivalent in mice, despite the high serum esterase activity in this species, and that an up to 10-fold reduction in total administered dose on the q3dX3 schedule is feasible by this approach.

Animals↗

PubMed1984

Methotrexate analogues. 21. Divergent influence of alkyl chain length on the dihydrofolate reductase affinity and cytotoxicity of methotrexate monoesters.

n-Octyl, n-dodecyl, and n-hexadecyl alpha- and gamma-esters of methotrexate (MTX) were compared with the previously described alpha- and gamma-n-butyl esters and with MTX as inhibitors of dihydrofolate reductase (DHFR) and human leukemic lymphoblasts (CEM cells) in culture. The overall order of activity in both test systems was MTX greater than MTX gamma-esters greater than MTX alpha-esters. In the DHFR assay the activity of the alpha-esters followed the order C4 greater than C8 congruent to C12 greater than C16, whereas for the gamma-esters this order was C4 congruent to C8 greater than C12 greater than C16. On the other hand, the order of cytotoxic activity in culture in both series was C16 greater than C12 greater than C8 greater than C4. Increasing the alkyl chain length in the ester moiety therefore decreases DHFR affinity but increases cytotoxicity. The most potent member of the compounds tested was the gamma-n-hexadecyl ester, whose IC50 against CEM cells was 0.11 microM as compared with 0.025 microM for MTX. In a comparison of the effect of treatment with the gamma-n-hexadecyl ester (10(-5) M, 1 h) on DNA synthesis in CEM and CEM/MTX cells, the latter of which are 120-fold resistant to MTX by virtue of a transport defect, the ester produced only 4-fold less inhibition in the resistant line than in the parental line. These results suggest possible use of this compound or related derivatives in the treatment of MTX-resistant tumors with impaired transport.

Animals↗

PubMed1984

Methotrexate analogues. 23. Synthesis, dihydrofolate reductase affinity, cytotoxicity, and in vivo antitumor activity of some putative degradation products of methotrexate-poly(L-lysine) conjugates.

Derivatives of methotrexate (MTX) in which the gamma-carboxyl group is joined to the epsilon-amino group of L-lysine, L-lysyl-L-lysine, or L-lysyl-L-lysyl-L-lysine, respectively, were prepared for evaluation of their dihydrofolate reductase (DHFR) affinity, their ability to retard cell growth in culture, and their antitumor activity in vivo. These small lysine derivatives of MTX are of interest as putative breakdown products of MTX-poly(L-lysine). Inhibition of DHFR in a cell-free assay was decreased only 3-fold relative to MTX, indicating that gamma-substitution by up to three lysines is well tolerated for binding. On the other hand, toxicity toward L1210 murine leukemia cells in culture decreased up to 120-fold relative to MTX as the lysines increased in number from one to three, suggesting that uptake across the cell membrane becomes difficult when positively charged lysines are at the gamma-position. Growth inhibition of H35 rat hepatoma cells was decreased 40- to 60-fold relative to MTX, but in H35R0.3 cells, which have normal DHFR content but are 180-fold MTX resistant by virtue of a transport defect, the lysine derivatives were only 3- to 7-fold less toxic than MTX. When the adducts were given to L1210 leukemic mice by twice-daily injection for 10 days, an increase in life span (ILS) of 80-100% was observed at 40 mg/kg (equivalent to 20-30 mg/kg of MTX). MTX itself, on the same schedule, gave a 100% ILS at 0.5 mg/kg. The low in vivo activity of the mono-, di-, and trilysine adducts suggests minimal systemic hydrolysis to free MTX.

Animals↗

PubMed1987

Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate.

Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.

Acetates↗

PubMed1988

Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.

Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.

Amino Acids, Dicarboxylic↗

PubMed2000

Methotrexate esters of poly(ethylene oxide)-block-poly(2-hydroxyethyl-L-aspartamide). Part I: Effects of the level of methotrexate conjugation on the stability of micelles and on drug release.

PURPOSE: To study the effects of hydrophobicity of the micelle-forming block copolymeric drug conjugate, methotrexate (MTX) esters of poly(ethylene oxide)-block-poly(2-hydroxyethyl-L-aspartamide) (MTX esters of PEO-b-PHEA), on the stability of micelles and on drug release. METHODS: MTX esters of PEO-b-PHEA with three levels of MTX conjugation were synthesized. Size distribution of the micelles was measured by dynamic light scattering (DLS). The critical micelle concentration (CMC) was determined by a light scattering study. Size exclusion high performance liquid chromatography (SEC-HPLC) was used to study the equilibrium between unimers and micelles, and release of MTX at pH 7.4. RESULTS: MTX esters of PEO-b-PHEA with MTX substitution of 7.4%, 22%, and 54% were prepared. The conjugates formed micelles based on DLS. The stability of the micelles correlated with the level of MTX conjugation. The conjugate with 54% MTX had a lower CMC (0.019 mg/mL) than the conjugates with 22% MTX (0.081 mg/mL) or 7.4% MTX (0.14 mg/mL). Micelle dissociation was significantly slower for the conjugate with 54% MTX than that with 22% and 7.4% MTX. Slower release of MTX from the micelles was also observed for the conjugate with the higher MTX attachment. CONCLUSIONS: MTX esters of PEO-b-PHEA can be structurally modulated by varying the degree of MTX substitution, which in turn changes the hydrophobicity of the conjugate, thereby modifying micelle stability and controlling drug release.

Antimetabolites, Antineoplastic↗

PubMed1990

A bispecific monoclonal antibody against methotrexate and a human tumour associated antigen augments cytotoxicity of methotrexate-carrier conjugate.

A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells.

Antibodies, Monoclonal↗

PubMed1983

Methotrexate cerebrospinal fluid and serum concentrations after intermediate-dose methotrexate infusion.

Twenty-nine children with acute lymphocytic leukemia were given 24-hr infusions of intermediate-dose methotrexate (MTX, 1000 mg/m2) with and without intrathecal (IT) MTX (12 mg/m2), followed by leucovorin rescue. There was substantial interpatient variability in MTX systemic clearance (98.3 +/- 51 ml/min/m2), inducing total steady-state serum MTX concentrations ranging from 5.4 to 33.7 microM. The cerebrospinal fluid (CSF) concentration at the end of the infusion was 0.27 (+/- 0.1) microM when no IT-MTX was given and correlated with total steady-state (24-hr) serum concentration of MTX. By stepwise regression, the CSF MTX concentration correlated better with the nonprotein bound (free) steady-state serum MTX concentration (r = 0.66, P less than 0.01) than with total steady-state serum MTX concentration. Mean CSF: serum MTX concentration ratio was 0.023 (+/- 0.04) when no IT MTX was given. When an IT MTX dose (12 mg/m2) was given at the start of the MTX infusion, the steady-state CSF MTX concentration was 1.1 (+/- 0.4) microM, leading to a mean CSF: serum ratio of 0.073 (+/- 0.05). Despite 7-hydroxy-MTX serum concentrations exceeding MTX concentrations immediately after infusion, 7-hydroxy-MTX was not detectable in CSF of most patients (21 of 29), and was less than 50% of the concurrent MTX concentration when detectable. These data establish the substantial interpatient variability in CSF distribution of MTX after intermediate-dose MTX infusions and establish a significant correlation between steady-state free concentration of MTX in serum and CSF MTX concentration.

Child↗

PubMed2003

The combination of calcipotriol and methotrexate compared with methotrexate and vehicle in psoriasis: results of a multicentre placebo-controlled randomized trial.

BACKGROUND: A multicentre, randomized, double-blind, vehicle-controlled, parallel-group study was carried out to study the effect of the addition of calcipotriol ointment to methotrexate (MTX) therapy in patients with psoriasis vulgaris. OBJECTIVES: To investigate whether the addition of calcipotriol to treatment with MTX has an MTX-sparing effect, and whether the combination of treatments is safe. Additionally, to compare the effect of calcipotriol or vehicle on the duration of the relapse-free interval after cessation of MTX. METHODS: Patients on maintenance therapy with MTX with controlled psoriasis were selected. The study was divided into three phases: (i) an MTX-free phase with double-blind treatment with either calcipotriol ointment or vehicle; (ii) an MTX titration phase with open MTX treatment and additional double-blind treatment with either calcipotriol or vehicle until target response; and (iii) follow-up phase: in a group of 97 patients, psoriasis was assessed using the modified psoriasis severity score, patients' assessment and safety parameters were monitored as well. RESULTS: The combined use of calcipotriol with MTX resulted in an MTX-sparing effect of 3.4 mg week-1 (phase (II) and 2.6 mg week-1 (phase I and II taken together), while still maintaining efficacy. Calcipotriol treatment increased the time to relapse of psoriasis following discontinuation of MTX: 113 days vs. 35 days. A decrease in aspartate aminotransferase and alanine aminotransferase was seen during the study of 8% (calcipotriol) and 12% (vehicle). CONCLUSIONS: The combination of calcipotriol and MTX was safe and well tolerated. The combination resulted in lower cumulative dosages of MTX compared with MTX and vehicle. Therefore the risk of side-effects is substantially decreased.

Adult↗

PubMed1999

Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression.

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-gamma production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-alpha gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-alpha production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.

Animals↗

PubMed1996

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Erythrocyte-methotrexate and disease activity in children treated with oral methotrexate for juvenile chronic arthritis.

The concentration of methotrexate (MTX) in erythrocytes (E-MTX) was measured twice with three months interval in 21 children suffering from juvenile chronic arthritis (JCA). At the same time joint score, visual analogue scale (VAS), and laboratory parameters (CRP, WBC, PMNs, and ALAT) were obtained. There was only a weak insignificant correlation between the dose of MTX/m2 and E-MTX (r=0.24, p=0.11). No significant relations between the clinical or laboratory parameters and E-MTX was found. However, ALAT above normal range was associated with a lower dose of MTX (p=0.02) and lower VAS (p=0.02), indicating that toxicity may be associated with less articular discomfort. At present we consider routine determination of E-MTX in children with JCA of limited value.

Administration, Oral↗

PubMed1987

Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow.

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.

Animals↗

PubMed1985

Dosage predictions in high-dose methotrexate infusions. Part 2: Bayesian estimation of methotrexate clearance.

Population pharmacokinetics of methotrexate (MTX) was evaluated from intravenous test-dose (TD) data (n = 20 corresponding to 174 measured samples). Bayesian prediction of MTX clearance from TD experiments combining population data with measured levels (at times 0.5 and 6 h) was found to be feasible in routine situations with good performance (root mean squared error : rmse (precision) = 1.14 1.h-1 (11.2%) and mean error : me (bias) = 0.06 1.h-1 (NS) relatively to weighted least-square estimates, n = 50). The precision of Bayesian prediction was comparable to that of the model independent which is used in routine practice and involves 9 measured levels over 30 h, (rmse = 1.35 1.h-1 (10.9%), n = 50). However, the routine method presented a significative bias (me = -0.81 1.h-1, n = 50).

Humans↗

PubMed1987

Folate and methotrexate polyglutamate tissue levels in rhesus monkeys following chronic low-dose methotrexate.

Methotrexate (MTX), a mainstay in the treatment of acute lymphoblastic leukemia, is associated with both hepatic and neurologic toxicity. Like a folate, MTX is metabolized to polyglutamated derivatives (MTXGlun) with long intracellular half-lives. These metabolites may contribute to MTX toxicity through a direct effect on cellular metabolism or indirectly through a perturbation of folate homeostasis. To better define the effects of chronic MTX treatment, tissue levels of MTX, MTXGlun, and folate were measured in three monkeys treated with weekly intramuscular MTX for 1 year. Greater than 80% of the total tissue MTX found was in the form of polyglutamated derivatives. Most of these derivatives were MTXGlu3-5 but Glu6-7 were easily detectable. Total tissue folates were measured in liver, kidney, brain and testis with MTX treated animals having a 90% loss of total folate in brain tissue. This is of special interest since inborn errors of folate metabolism are often associated with severe neurologic abnormalities.

Animals↗