Medication Error- A Case Report of Misadventure with Methotrexate.

Methotrexate is an antimetabolite drug with antineoplastic and immunomodulatory properties, useful as an antineoplastic agent in various haematological and solid tumours. MTX toxicity can occur because of accidental ingestion/overdose by the patient or because of prescription error. The toxic effects manifest as severe mucositis or as organ damage (bone marrow depression, renal/hepatic injury). The toxicity usually results from parenteral overdose or repeated chronic drug ingestion. Acute high dose ingestion does not result in MTX toxicity because of saturable absorption kinetics. We present a case of MTX toxicity occurring as a result of prescription error resulting in repeat daily dosing of the drug, and the challenges associated with the management of the same, in a patient with multiple comorbidities. The present case emphasizes on a note of caution on the part of the prescriber and the suggestions regarding the measures which can be taken to avoid MTX toxicity. Keywords: drug overdose; Methotrexate; mucositis; pancytopenia.

Is post exposure prevention of teratogenic damage possible: Studies on diabetes, valproic acid, alcohol and anti folates in pregnancy: Animal studies with reflection to human.

We discuss the possibilities to prevent the post-exposure teratogenic effects of several teratogens: valproic acid (VPA), diabetes and alcohol. Co-administration of folic acid with VPA reduced the rate of Neural Tube Defects (NTD) and other anomalies in rodents, but apparently not in pregnant women. Antioxidants or the methyl donor S-adenosyl methionine prevented Autism Spectrum Disorder (ASD) like behavior in mice and rats. In vivo and in vitro studies demonstrated that antioxidants, arachidonic acid, myoinositol and nutritional agents may prevent diabetes-embryopathy. Prevention of alcohol-induced embryonic and fetal injuries and neurodevelopmental deficits was achieved by supplementation of zinc, choline, vasoactive intestinal proteins (VIP related peptides), antioxidants and folic acid. While the animal research described in this review is indicative of possible preventions of the different teratogenic effects, this is not yet the focus in human research. Future research should promote further knowledge where our current understanding is the vaguest, human prevention.

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development.

Virgin coconut oil protects against liver damage in albino rats challenged with the anti-folate combination, trimethoprim-sulfamethoxazole.

BACKGROUND: Trimethoprim-sulfamethoxazole (TMP-SMX) is a broad-spectrum antibiotic. However, its use is associated with toxic reactions. Virgin coconut oil (VCO), derived from coconut, has been widely used throughout history for its medicinal value. The aim of this study was to investigate the beneficial actions of VCO against TMP-SMX-induced alterations in serum biochemical end points. METHODS: Twenty rats were divided into four groups. Group 1 (control) received no drug, whereas group 2 received TMP-SMX (8/40 mg/kg) twice daily for 7 days. Group 3 was administered coconut oil at a dose of 600 mg/kg body weight per day. The last group was treated with TMP-SMX (8/40 mg/kg) and coconut oil (600 mg/kg) simultaneously. Blood samples were collected from all groups on the 8th day of the experiment for measurement of serum biochemical parameters. Organ weights and coefficients were also evaluated. RESULTS: TMP-SMX caused a significant (p<0.05) increase in the levels of serum total bilirubin, lactate dehydrogenase, and alkaline phosphatase by 192%, 67%, and 41%, respectively, relative to controls. This was followed by a significant reduction in triglyceride and relative kidney weight by 40% and 7%, respectively. There were no significant differences (p>0.05) in the activities of serum aminotransferases, total acid phosphatase, γ-glutamyl transferase, uric acid, cholesterol, albumin, and urea levels. Supplementation of VCO ameliorated TMP-SMX-induced effects by restoring the levels of total bilirubin, alkaline phospahatase, and lactate dehydrogenase. CONCLUSIONS: The results of this study demonstrate that the active components of coconut oil had protective effects against the toxic effects induced by TMP-SMX administration, especially in the liver of rats.

Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria.

Methotrexate (MTX) is a folic acid antagonist widely used as a cytotoxic chemotherapeutic agent for leukemia and other malignancies. The purpose of this study was to investigate the damage caused by MTX on liver mitochondria and its protection by using antioxidant properties of lipoic acid. MTX substantially affects mitochondrial function by reducing glutathione levels leading to disturbances in antioxidant enzyme defense system. Lipoic acid occurs naturally in mitochondria as a coenzyme. In various studies lipoic acid has been convincingly shown to exhibit an antioxidant role when supplemented exogenously. We studied the effect of lipoic acid pre-treatment on the toxicity of MTX in mouse liver mitochondria focusing specifically on the oxidative stress. MTX caused a significant rise in the mitochondrial lipid peroxidation (LPO), protein carbonyl (PC) content and superoxide radical generation. It also affected the mitochondrial thiol profile. Pre-treatment of mice with lipoic acid (35 mg/kg) markedly lowered mitochondrial LPO, PC content and superoxide radical generation. It also restored decreased enzymatic and non-enzymatic antioxidants of mitochondria. It is suggested that lipoic acid has a potential role in suppressing MTX-induced mitochondrial toxicity, and it affords protection either by reversing the decline of antioxidants or by the directly scavenging the free radicals.

Potent protective effect of apricot and beta-carotene on methotrexate-induced intestinal oxidative damage in rats.

Several studies have well confirmed the contribution of oxidative stress in the pathogenesis of methotrexate (MTX)-induced damage in the small intestine. Many agents have been tried experimentally to reduce or inhibit the oxidative stress. To our knowledge, there is no study about apricot consumption on the MTX-induced damage in the small intestine. The aim of this study was to determine the possible protective effects of apricot and beta-carotene on MTX-induced intestinal damage in rats. The rats were randomly divided into seven groups as follows; I-control group; II-apricot group; III-beta-carotene group; IV-MTX group; V-apricot+MTX group; VI-beta-carotene+MTX group and VII-apricot+beta-carotene+MTX group. In the MTX group; fusion and shortening in the villus, epithelial desquamation, crypt loss, inflammatory cell infiltration in the lamina propria, goblet cell depletion and microvillar damage were observed in the small intestine. Parallel to histological results, malondialdehyde (MDA) content and myeloperoxidase (MPO) activity were found to be increased, whereas superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP-x) activities and glutathione (GSH) content were decreased in the MTX group. However, single or combined application of apricot and beta-carotene ameliorated all of these hazardous effects in antioxidant system in MTX-treated groups. In conclusion, our results demonstrate that apricot and/or beta-carotene treatment may protect the impairment of oxidative stress and ameliorate MTX-induced intestine damage at biochemical and histological levels.

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates.

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.

Folinic acid protects against suppression of growth by methotrexate in mice.

The objective of this study was to investigate whether folinic acid supplementation would protect young mice against suppression of growth by methotrexate (MTX). Four equal groups of Balb/c young male mice (5 animals in each group; mean+/-SD body weight 9.64+/-0.85 g, in their rapid growth phase) were subjected to the following drug treatment: One group was given MTX (3.5 mg/kg body weight) intraperitoneally on every 2nd day, another received folinic acid (7.0 mg/kg body weight) intraperitoneally every 2nd day. The third group was given both of these drugs (MTX on every 2nd day and folinic acid 8 h post-MTX injection). The fourth group was injected with physiological saline every other day to serve as a control group. Total body weight, food and water consumption by animals in each group were monitored every second day for a period of 3 weeks. After this period mice were sacrificed and liver, spleen and kidneys were excised, weighed and analyzed for MTX and dihydrofolate reductase activity. A small segment of the proximal part of small intestine and small pieces of liver and kidney were also removed to study morphological changes. Compared to the groups, which received folinic acid alone, folinic acid plus MTX or physiological saline, mean increase in body weight (6.8+/-0.8 g) of mice over a period of 3 weeks was minimal in the group receiving MTX alone (one-way ANOVA p=0.0001). The mean weights of liver and kidney in this group receiving MTX alone were also found to be significantly less than the mean weights of these organs in the 3 groups (p<0.001). The negative effect on growth of animals appears not only due to malabsorption but inhibition of pathway of de novo DNA synthesis may also be involved. This is supported by loss of villous pattern in small intestine of mice treated with MTX alone and increased accumulation of free MTX and decreased dihydrofolate reductase in the liver of the group receiving MTX alone as compared with the group receiving MTX plus folinic acid. The data indicate that the administration of folinic acid protects mice against suppression of growth by MTX. On the basis of these observations it can be deduced that patients suffering from juvenile rheumatoid arthritis or acute lymphoblastic leukaemia receiving MTX over a long period of time might be at a risk of experiencing short-term suppression of growth, however they could benefit from supplementation with folinic acid.

Phenoxypropoxybiguanides, prodrugs of DHFR-inhibiting diaminotriazine antimalarials.

A total of 34 analogues of the biguanide PS-15 (5s), a prodrug of the diaminotriazine WR-99210 (8s), have been prepared. Several of them, such as 5b (PS-33) and 5m (PS-26), maintain or exceed the in vivo activity of PS-15 while not requiring the use of highly regulated starting materials. The putative diaminotriazine metabolites of these new analogues (compounds 8) have also been prepared and shown to maintain the activity against resistant P. falciparum strains. The structure-activity relationships of biguanides 5 and putative metabolites 8 are discussed.

Folic acid and human malformations: a summary and evaluation.

A large body of evidence gathered over the past 30 or more years has led to the firmly established belief that deficiency of the vitamin folic acid is a cause of congenital malformations of neural tube closure. Beginning with studies showing folic acid levels of mothers of children with such defects to be low, this belief has been solidified by epidemiologic studies revealing that this consequence is prevented by maternal supplements of the vitamin from early pregnancy. The present article reviews this evidence for the purpose of examining the claims of the efficacy of folic acid in this respect. This seems to be an advantageous moment to do so, because no clear impact of folic acid supplementation and fortification on the prevalence of neural tube defects has as yet been documented; and furthermore a pause seems to have been reached in such studies. It is felt that a historical, i.e. a chronologic approach will best describe the findings, and therefore they will be considered as they unfolded.

Efficacy of folinic acid in reducing methotrexate toxicity in juvenile idiopathic arthritis.

OBJECTIVE: To investigate the efficacy of folinic acid in reducing the side effects associated with methotrexate (MTX) therapy in children with juvenile idiopathic arthritis (JIA) and to determine whether folate supplementation may reduce the benefit of MTX administration. METHODS: This was a retrospective, non-controlled study. Inclusion criteria were: 1) diagnosis of JIA according to the Durban 1997 criteria; 2) treatment with low to intermediate doses of MTX (10-20 mg/m2/week) as the sole second-line agent for at least 6 mos.; and 3) supplementation with folinic acid (2.5-7.5 mg) in a single weekly dose 24 hrs after MTX administration. All patients were started on folinic acid only after the development of a side effect. Exclusion criteria were: treatment with higher doses of MTX (> 20 mg/m2/week). The outcomes investigated were: hepatotoxicity (liver transaminase increase), gastrointestinal toxicity, disease flare, and clinical remission. The number of episodes per patient-year of MTX treatment of each outcome before and after folinic acid supplementation was compared by the Wilcoxon matched pairs test. RESULTS: A total of 43 children with JIA were included in the study. The mean duration of treatment before and after folinic acid supplementation was 1.1 years and 1.8 years, respectively. After the start of folinic acid supplementation, the mean number of episodes per patient-year of hepatotoxicity and gastrointestinal toxicity decreased from 2.30 to 0.32 (p < 0.001) and from 1.09 to 0.29 (p = 0.002), respectively. The mean number of disease flares and clinical remissions per patient-year did not change significantly. CONCLUSION: In our JIA patients, folinic acid supplementation resulted in a significant reduction in the most common side effects of MTX, without affecting the clinical efficacy of the drug.

A soy-derived antiapoptotic fraction decreases methotrexate toxicity in the gastrointestinal tract of the rat.

The ability of a soy-derived antiapoptotic fraction to inhibit methotrexate-induced gastrointestinal toxicity was examined. Male Sprague-Dawley rats treated with methotrexate were fed diets containing casein as a sole protein source or diets supplemented with a protein-phospholipid fraction isolated from soy flour. This soy fraction has also been shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in the mouse embryonic C3H10T1/2 cell. Rats that received high doses of the soy-derived antiapoptotic fraction-supplemented diets experienced significantly less weight loss and diarrhea and better maintained their pretreatment appetite.

Functional aspects of membrane folate receptors in human breast cancer cells with transport-related resistance to methotrexate.

Two methotrexate (MTX)-resistant human breast-cancer cell lines with impaired transport via the reduced folate carrier (RFC), one established in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231), were adapted to grow in medium containing 2 nM folic acid. This induced the expression of previously undetectable membrane folate receptors (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Polymerase chain reaction (PCR) quantitation revealed that MFR messenger-RNA levels of the isoform first described in human nasopharyngeal carcinoma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)-ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth-inhibitory effects of five different antifolates with varying affinities for MFR: N10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-dideazatetra-hydrofolic acid (DDATHF) > N-5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-methyl) -N-methyl-amino]-2-theonyl}-glutamic acid (ZD1694) >> MTX > edatrexate (EDX). Expression of MFR only slightly decreased the resistant phenotype for MTX, EDX, and ZD1694, suggesting that these drugs are not transported intracellularly to cytotoxic concentrations at these levels of MFR expression. On the other hand, both cell lines became from at least 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. These sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affinity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cells with the highest level of MFR expression (MTX(R)-ZR-75-1). These results illustrate that the efficacy of MFR in mediating antifolate transport and cytotoxicity depends on their affinity for the folate antagonist, their degree of expression, and the levels of competing folates.

High-dose 7-hydromethotrexate: acute toxicity and lethality in a rat model.

To elucidate mechanisms for methotrexate (MTX)-induced renal and hepatic toxicity, we investigated the acute effects of bolus plus continuous infusion of up to 0.4 g/kg 7-hydroxymethotrexate (7-OH-MTX) in the rat. We demonstrate for the first time in any species the occurrence of acute lethal toxicity within a few hours after 7-OH-MTX administration. Serum concentrations of 7-OH-MTX measured at the time of death were 1.4 mM (mean), about one-half of those achieved in some patients after infusion of high-dose MTX (HD-MTX) in the clinic. The data suggest an approximate LD50 (the dose lethal to 50% of the study population) of 0.3 g/kg and a steep dose/lethality curve for 7-OH-MTX. Moreover, acute renal and hepatic toxicity occurred as evidenced by severe morphological findings and increased serum levels of creatinine and liver transaminases. In all rats subjected to continuous infusion of 7-OH-MTX, yellow microscopic precipitations were apparent in the kidney tubules. Crystallization was also seen in bile ducts of the liver in some of the rats. These results further support that the formation of 7-OH-MTX is disadvantageous and that reported attempts to prevent its formation during MTX treatment are warranted.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat.

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 microM, and concentrations of 7-OH-MTX declined triphasically, showing a t1/2 alpha value of 2.45 min, a t1/2 beta value of 30.5 min, and a terminal half-life (t1/2 gamma) of 240 min. The total clearance value was 14.5 ml min-1 kg, and the postdistributional volume of distribution (V beta) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

New thiophene substituted 10-deazaaminopterins: synthesis and biological evaluation.

Analogues of 10-deazaaminopterin (10-DAM) and 4-amino-4-deoxy-10-deazapteroyl-gamma-methylene glutamic acid (MDAM) in which the benzene ring was replaced with a thiophene ring have been synthesized and evaluated for their antitumor activity. These analogues were N-([5-(2,4-diamino-6-pteridinyl)ethyl]-2-thenoyl)-L- glutamic acid (1) and N-([5-(2,4-diamino-6-pteridinyl)ethyl]-2-thenoyl)-gamma-meth ylene glutamic acid (2).

Cisplatin and dichloromethotrexate: a pharmacologically rational combination.

Dichloromethotrexate (DCM), a dihalogenated analog of methotrexate (MTX), is a potent classic folate antagonist that is metabolized and excreted by the liver. It is not nephrotoxic nor are its pharmacokinetics significantly affected by changes in renal function. Therefore, the feasibility of administering a maximum tolerated dose of DCM in combination with high-dose ciplatin (CDDP) was investigated in patients with head and neck, bladder, or cervical cancer--three epithelial carcinomas known to be responsive to single-agent CDDP and MTX. The overall response rates in 24 evaluable patients with head and neck cancer, 21 evaluable patients with bladder cancer, and 22 evaluable patients with cervical cancer were 54%, 57%, and 50%, respectively. Complete response rates of 25%, 19%, and 36%, respectively, were observed. DCM can be administered on a weekly schedule at a maximum tolerated dose in combination with CDDP with tolerable side effects and without significant nephrotoxicity.

Trimetrexate: a new antifol entering clinical trials.

Trimetrexate, a 2,4-diaminoquinazoline derivative, is a new antifol recently introduced into clinical trials. It differs from methotrexate principally in its transport (not carrier-mediated), and its intracellular retention (not polyglutamylated). Trimetrexate is active against tumors which are methotrexate-resistant on the basis of impaired transport, and has a broader range of antitumor activity in preclinical models. Animal studies predict toxicity principally to the central nervous system, gastrointestinal tract and bone marrow.