Toxic Effects Of Methotrexate Research Articles

Therapeutic potential of diosgenin against methotrexate-induced testicular damage in the rat.

This study evaluated diosgenin effects on methotrexate-induced testicular injury in the rats. A single dose of methotrexate (MTX) (20mg/kg, i.p) was administered, followed by two weeks of diosgenin treatment via gavage starting one day before methotrexate injection. Testicular damage was evaluated through histological examination of seminiferous tubules, as well as analysis of serum testosterone level, oxidative stress and inflammation biomarkers, and antioxidant levels. The results of this study showed that in the MTX-exposed group, oxidative stress indices of malondialdehyde (MDA), reactive oxygen species (ROS), nitrite and indices of inflammation consisting of tumor necrosis factor α (TNFα), and interleukin 6 (IL-6) have a significant increase compared to the control group. Additionally, reductions were observed in antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). In addition, testosterone level decreased and signs of testicular damage were observed in the MTX group. Conversely, in the group treated with diosgenin alongside MTX at a dosage of 50mg/kg, there was a significant decrease in oxidative stress markers (MDA, ROS, nitrite) and inflammatory markers (TNFα and IL-6). Moreover, there was a significant increase in the levels of antioxidant enzymes (SOD, CAT, and GSH). Diosgenin appears to have the potential to protect testicular tissue from damage caused by the toxic effects of MTX through the reduction of oxidative stress and inflammation.

Influence of ABCB1 3435C>T Polymorphism on Methotrexate Safety in Patients with Psoriasis — A Secondary Publication

Background: Methotrexate is a highly effective systemic treatment for moderate to severe psoriasis, but drug toxicity may limit its use. Recent evidence suggests that it is necessary to take into account the individual characteristics of methotrexate pharmacokinetics, which are determined by the presence of polymorphisms of genes encoding methotrexate carrier proteins, to predict the risk of methotrexate-induced toxicity. Aim: The research aims to assess the associations of ABCB1 rs1045642 (3435C>T) polymorphism with methotrexate safety for patients with moderate and severe psoriasis. Methods: The study included 75 psoriasis patients treated with methotrexate with 21 days of follow-up duration. Data on adverse drug reactions (ADR) were collected using a clinically structured questionnaire, and complete and biochemical blood tests, and urinalysis were performed. The severity of ADR was assessed using visual analog scales and the CTCAE toxicity scale. The severity of gastrointestinal ADR was assessed using the GSRS questionnaire. Genotyping was carried out by real-time PCR. Results: Gastrointestinal toxicity was detected in 38 patients (50.67%). The mean GSRS score was 7.97 ± 9.18. Analysis of differences in the ADR incidence showed the presence of statistically significant differences in the frequency of ADR in the gastrointestinal tract: the toxic effect of methotrexate was more often observed in carriers of the T allele of the ABCB1 rs1045642 polymorphism (3435C>T), (CC: 2 (14.3%), TC: 18 (52.9%), TT: 18 (66.7%), P = 0.006). Binomial regression demonstrated the presence of a statistically significant effect of the rs1045642 single-nucleotide polymorphism of the ABCB1 gene on the incidence of ADR from the gastrointestinal tract (OR = 8.64, P = 0.008). Conclusion: An association of ABCB1 rs1045642 single-nucleotide polymorphism with the safety of methotrexate therapy in patients with moderate and severe psoriasis was revealed. The data obtained can be used to personalize the prescription of methotrexate to psoriasis patients.

The Effect of Alpha-Lipoic Acid against Methotrexate on Testicular Damage in Rats

Aim: The toxic effects of methotrexate, a chemotherapeutic, on the testicles is an important side effect. Methotrexate impairs spermatogenesis and fertility and causes oligospermia. In this study, we aimed to minimize the testicular toxicity, those being the side effects of methotrexate, by using the probable protective effects of α-lipoic acid, a potent antioxidant.
 Materials and Methods: Twenty-eight male Sprague Dawley rats that we employed in this research were separated into three groups as control (0.09% PS) (n=8), methotrexate (20 mg/kg) (n= 10), and methotrexate (20 mg/kg) + α-lipoic acid (100 mg/kg) (n= 10). We performed a histochemical analysis on the testicular tissue of rats using hematoxylin-eosin and Masson’s trichrome. We performed an immunohistochemical analysis using inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) primer ab.
 Results: The histochemical evaluation revealed a significant decrease in the methotrexate-induced testicular toxicity in the α-lipoic acid-treated groups. On the other hand, TNF-α and iNOS immunostaining results were also observed to support these results.
 Conclusion: The treatment use of α-lipoic acid succeeded in protecting against methotrexate-induced testicular damage through an α-lipoic acid-mediated antioxidant and anti-inflammatory mechanisms. α-lipoic acid can be used in combination with methotrexate as a protector against side effects during anticancer therapy. In the present study, it was shown that α-lipoic acid can be used in combination with methotrexate as a protector against side effects during anticancer treatment.

Influence of <i>ABCB1</i> <i>3435C>T</i> polymorphism on methotrexate safety in patients with psoriasis

Background. Methotrexate is a highly effective systemic treatment for moderate to severe psoriasis, but drug toxicity may limit its use. Recent evidence suggests that it is necessary to take into account the individual characteristics of methotrexate pharmacokinetics, which are determined by the presence of polymorphisms of genes encoding methotrexate carrier proteins, to predict the risk of methotrexate-induced toxicity.
 Aims. The research aim is the assessment of associations of ABCB1 rs1045642 (3435CT) polymorphism with methotrexate safety for the patients with moderate and severe forms of psoriasis.
 Materials and methods. The study included 75 patients diagnosed with psoriasis treated with methotrexate during 21 day. Data on adverse drug reactions were collected using a clinically structured questionnaire, complete and biochemical blood tests, and urinalysis. The severity of adverse drug reactions was assessed using visual analog scales and the CTCAE toxicity scale. The severity of gastrointestinal ADR was assessed using the GSRS questionnaire. Genotyping was carried out by real-time PCR.
 Results. The gastrointestinal toxicity was detected in 38 patients (50.67%). The mean GSRS score was 7.97 9.18. Analysis of differences in the incidence of adverse drug reactions showed the presence of statistically significant differences in the frequency of adverse drug reactions in the gastrointestinal tract: the toxic effect of methotrexate was more often observed in carriers of the T allele of the ABCB1 rs1045642 polymorphism (3435CT), (CC 2 (14.3%), TC 18 (52.9%), TT 18 (66.7%), p = 0.006). Binomial regression demonstrated the presence of a statistically significant effect of the rs1045642 SNP polymorphism of the ABCB1 gene on the incidence of ADR from the gastrointestinal tract (OR = 8.64, p = 0.008).
 Conclusions. An association of ABCB1 rs1045642 SNP with the safety of gastrointestinal methotrexate therapy in patients with moderate and severe forms of psoriasis was revealed. The data obtained can be used to personalize the prescription of methotrexate to patients with psoriasis.

In vivo immunoloprotective potentials of Plantago lanceolata aqueous extract on methotrexate induced albino male mice

This study aimed to evaluate the immunological potential of Plantago lanceolata through determination of (total and absolute count of white blood cell, total count of red blood cell and total count of hemoglobin). The results indicated the ability of plant extract to modulate toxic effect of methotrexate on albino mice by enhancing immunity through all tested parameters All this effect due to the presence of chemical active constituents of plant especially (flavonoid and alkaloids).

ORAL ULCERS CAUSED BY THE USE OF METHOTREXATE IN A PATIENT WITH RHEUMATOID ARTHRITIS

Methotrexate is a chemotherapy originally used in oncology that is also applied in several nononcologic diseases, such as rheumatoid arthritis, because it has an anti-inflammatory effect in low doses. A 68-year-old leucoderm man attended our service with complaint of oral sores for 1 month. As a personal background he had rheumatoid arthritis, diabetes, hypertension, and hypothyroidism. The patient said the ulcers began to appear after the dose of methotrexate had been changed. Clinical history and anamnesis as well as a conversation with his physician led to the conclusion that a possible methotrexate toxicity was related to the oral ulcers despite the use of the medication in low doses (25 mg weekly). Extensive sore of the oral mucosa, especially in the labial lips region, was observed with a yellowish membrane. This case report aims to demonstrate that the toxic effects of methotrexate are seen even in low doses.

Toxic Effects of Methotrexate on Rat Kidney Recovered by Crocin as a Consequence of Antioxidant Activity and Lipid Peroxidation Prevention

Background:The application of MTX as a chemotherapy agent and immune system suppressant has various side effects. Crocin, a xanthine derivative plant, has many therapeutic benefits. This study was planned to assess the effect of crocin on renal toxicity of MTX in a rat model. Methods:Forty eight rats were divided randomly into eight groups (n = 6), which received normal saline, MTX, crocin, and MTX + crocin for 28 days intraperitoneally. The levels of oxidative stress in kidney and blood serum were measured, and the kidney was analyzed histologically.Results:MTX caused an enhancement in the levels of thiobarbituric acid reactive substances and biochemical marker (creatinine and BUN). Besides, a significant decrease was observed in tissue parameters and antioxidant capacity compared to the normal control group (p < 0.001). The crocin and crocin + MTX decreased the biochemical markers, the levels of thiobarbituric acid reactive species, and tissue parameters considerably at entire dose (12.5, 25, and 50 mg/kg) and enhanced the antioxidant capacity levels compared to the MTX group (p < 0.001). Conclusion:Administration of crocin improves the damage caused by MTX in rats. The crocin by the establishment of balance in the levels of antioxidant prevents the damage to the renal cell membrane, and subsequently the renal damage repairs.

IN VITRO STUDIES ON THE SYNERGISTIC EFFECT OF MORINDA CITRIFOLIA L. (NONI) AND METHOTREXATE ON CYTOTOXICITY OF HELA CELL LINES

Objective: Cancer remains to be one of the leading causes of death around the world. Modern targeted therapies have undeniably improved cancer patients’ survival, but search still continues for safer and more effective drugs. This work is an attempt to assess the effectiveness of cotreatment of aqueous fruit extract of Morinda citrifolia L. (Noni) on methotrexate (MTX)-induced cytotoxicity on HeLa cancer cell line.&#x0D; Methods: HeLa cells were treated with different concentrations of MTX and Noni alone and in combination for 24 and 48 h and studied for the cytotoxic effects by various approaches.&#x0D; Results: There was a dose-dependent inhibition of growth when treated with MTX in a dose range of 0.045–45.4 μg/ml and Noni for a dose range of 0.3–30 mg/ml for 48 h. The IC50 for MTX was 4.45 μg/ml (1 μM/ml), and for Noni, it was 8 mg/ml while the combination exhibited a more intensive growth inhibitory effect. Our results were confirmed with phase-contrast microscopy, whereby the number of viable cells decreased with an increase in the concentration of Noni. Cells were found to have rounded up and detached from the flask indicating apoptosis. Apoptosis in HeLa cell lines was further confirmed by DNA fragmentation and flow cytometry.&#x0D; Conclusion: It can be concluded that Noni may enhance MTX cytotoxicity by inducing apoptosis and cell cycle arrest. Hence, cotreatment with Noni may reduce the dosage of MTX necessary for inhibiting proliferation of malignant cells and hence decrease the toxic effects of MTX on the system.

THE USE OF NUTRIENT RESTRICTION IN COMBINATION WITH CHEMOTHERAPY IN LEWIS’ LUNG CARCINOMA

The aim of the study was to evaluate the effect of nutrient restriction in combination with chemotherapy with methotrexate on the development of Lewis lung carcinoma (LLC) in mice. Material and methods. LLC cells were inoculated into the thigh of adult C57BL/6j mice. Mice fasted twice, for 2 days each, with a day interval. Methotrexate (MTX) was used alone and in combination with nutrient restriction. The drug was administered intraperitoneally at the maximally tolerated dose. Treatment efficacy was evaluated by the tumor growth inhibition (TGI), the level of inhibition of lung metastases (IM) and the kinetic tumor growth index (GI). Results. TGI was 34 %, 27 % and 46 % in mice with nutrient restriction alone, MTX alone and with combination of nutrient restriction and MTX, respectively. IM index was 90% and 70% in mice treated with MTX alone and in combination with nutrient restriction, respectively. There was no difference in IM index between mice subjected to restricted feeding and mice in the control group. GI was 0.7 in mice with nutrient restriction and combination of nutrient restriction and MTX. GI was 0.81 in mice treated with MTX monotherapy. The toxic effect of MTX was manifested by weight loss in mice. Weigh loss by 13% and 17% was observed in mice subjected to nutrient restriction alone and in combination with MTX, respectively. Conclusion. MTX inhibited cancer cell growth, however, the therapeutic effect of MTX was leveled by toxic effect on mice. Prolonged caloric restriction inhibited primary tumor growth. However, after returning to a normal diet, tumor growth resumed quickly. The use of MTX in combination with caloric restriction increased its anti-tumor effect on the primary tumor, decreased its toxic effect, but reduced its anti-metastatic activity by 20 %.

The determination of the folic acid and its selected derivatives

Folic acid (FA) belongs to vitamins from group B. The most commonly used drug from a group of structural analogues of FA is methotrexate (MTX), showing antiproliferatative properties. Unfortunately, the action of MTX is not selective. Toxic effects of methotrexate may be partially abolished by the use of leucovorin (calcium salt of folinic acid, FNA) in conserving therapy. The aim of the presented paper was to develop a new analytical procedure using the capillary electrophoresis, allowing the separation and the quantitative marking of the folic acid, methotrexate and calcium salt of folinic acid. Optimal conditions for the electrophoretic separation were obtained for the following parameters: buffer - 25 mmol/L Na2B4O7,pH 9,76; voltage -30 kV, measurement temperature -30 °C, the introduction of a sample to the capillary - hydrodynamically 6 s (0,5 psi). These parameters allowed to obtain LOD 0,6 μM for FA, 0,65 μM for FNA and 0,7 μM for MTX.

Effect of Methotrexate on the Jejunum of Adult Albino Rat and the Possible Protective Role of Vitamin A: Histological and Immunohistochemical Study

Background: Methotrexate (MTX) is an antineoplastic drug that is widely used in the treatment of neoplasms. Patients undergoing MTX therapy have a variety of side effects affecting many important organs. Aim of the work: The aim of the present study was to detect the histological and the immunohistochemical adverse alterations that might occur in the jejunum of the adult albino rats following methotrexate therapy and to determine the possible protective role of vitamin A. Material and Methods: Seventy two adult male albino rats were divided into six equal groups. In all groups the animals were sacrificed 24 hours after the last injection. The jejunal specimens were examined using H& E and Masson’s trichrome stains and immunohistochemical examination to study the alkaline phosphatase activity using light microscopy. Morphometric study measuring villous height, crypt depth, number of goblet cells, percentage area of fibrosis and the optical density of alkaline phosphatase activity was done in all groups. Results: The methotrexate treated groups’ revealed different changes in the jejunum of rats. Distortion and cystic dilatation of the crypts, cellular shedding and dilated blood vessels were found. Morphometric study showed decreased villus height and goblet cells, increased the crypts depth. Immunohistochemical examination of the jejunal sections of the methotrexate treated groups showed weak reaction of alkaline phosphatase enzyme when compared with that of the control group. Conclusion: It can be concluded that the toxic effect of methotrexate on the jejunum of rats can be partially improved with the concomitant use of vitamin A.

Preparation and characterization of methotrexate-loaded microcapsules

Methotrexate (MTX) has toxic effect to healthy tissues. Microencapsulation coats particles with a functional coat to optimize storage stability and to modulate release. In the present study, a new MTX encapsulated microcapsules were synthesized for controlling MTX release. Controlled drug release provides releasing of efficient dose and prevent drug side effect to tissues and also protects MTX from oxygen, pH and other interactions. MTX was encapsulated through biocompatible hyaluronic acid (HA) and sodium alginate (SA) with an encapsulation system to reduce its toxicity and for controlled release. The microcapsules prepared by vibrating nozzle were cross-linked with SA, HA and calcium chloride. Nozzle diameter and MTX concentration were changed according to loaded MTX and encapsulation efficiency were determined using HPLC. For the reliability of the data, validation studies of the HPLC method were performed. The precision of the method was demonstrated using intra- and inter-day assay relative standard deviation (RSD) values which are less than 2% in all instances. For the characterization of microcapsules, particle size, drug loading and in vitro drug release studies were performed. Diameters of MTX-loaded microcapsules were acquired approximately 160, 400 and 800 µm. Surface morphology of encapsulated microcapsules were displayed with light microscope. Eighty-nine percent MTX encapsulation efficiencies were achieved. Encapsulated MTX microcapsules showed controlled release when compared to pure MTX. While powder MTX dissolved completely in 10 min in the dissolution medium, MTX release from encapsulated MTX microcapsules became 40 h in 0.1 M PBS pH 7.4, including NaCl. MTX release from MTX-loaded microcapsules was reached to 79%. Moreover, drug efficiency was examined in vitro cell culture tests. Viability of 5RP7 cells were decreased to 88.5% for 96 h. When MTX was given directly to 5RP7 cells, viability of 5RP7 cells was decreased to 49.7% for 96 h. Flow cytometry studies also showed that, MTX microcapsules induced apoptosis. The goal of this study is to provide controlled release of MTX and to reduce the toxic effect of MTX.

Glucarpidase: Treatment for toxic MTX concentrations

Glucarpidase: Treatment for toxic MTX concentrations

The future of folic acid antagonist therapy in rheumatoid arthritis

When, at the end of the last decade, the new and potent tumor necrosis factor (TNF ) inhibitors were approved for the treatment of rheumatoid arthritis (RA), the era of methotrexate (MTX) as the gold standard for treatment of this disease seemed to have come to an inevitable end. On the one hand, there were the new, intelligently designed and highly specific cytokine inhibitors, a result of the fast-growing knowledge about immunopathologic mechanisms of inflammation. On the other hand, there was MTX, a byproduct of oncology research from decades past. The exact mode of antiarthritic action of this antifolate drug in the complex system of inflammation could only be hypothesized. Now, several years later, MTX plays a more important role than ever as the basis of RA treatment. Due to its effectiveness and tolerability, MTX remains the drug of first choice in the treatment of RA. Moreover, in order to make the new biologic drugs as effective as possible, almost all of the new investigational drugs are tested in combination with MTX. The designs of these clinical trials mainly resemble the design of the original Anti–TNF Trial in RA with Concomitant Therapy (ATTRACT) study (1), in which MTX was given in combination with infliximab, at that time still with the purpose of inhibiting the formation of antibodies against the drug. Meanwhile, not only TNF inhibitors (2,3), but also many other new biologic drugs are given in combination with MTX because this strongly increases the magnitude and duration of the therapeutic response. Although there is now a growing number of new drugs available for the treatment of RA, it is widely accepted that the question is not whether a patient with RA should receive MTX or not, but rather, whether the patient should receive it with or without a biologic drug. Thus, the principle of folate antagonism seems to remain the foundation of immunomodulatory treatment of RA, both now and in the future. It should be emphasized that MTX is experiencing a comeback in RA clinical trials, although this drug has several pharmacologic disadvantages that one might think should be limiting its use. First, if given orally, MTX shows a wide variability of resorption, ranging in some studies from 28% to 88% (4). Second, although it is given only once a week, it has a short half-life in the circulation, consisting of only 5–8 hours (4). Therefore, it is thought that its antiarthritic effect might partly depend on the rate of polyglutamation, an intracellular transformation of MTX that results in intracellular persistence of the drug. The rate of this modification, which is possibly crucial for its therapeutic effect, has a marked interindividual variability (5) and is influenced by independent factors, such as estrogen, insulin, or dexamethasone (6). Finally, the intracellular uptake of the molecule and its pharmacologic actions on intracellular targets are characterized by a broad range of mechanisms (7). This makes differentiation between the mechanisms of the unwanted toxic effects of MTX and the wanted antiinflammatory effects difficult. Different approaches have been tried in an attempt to overcome the disadvantageous properties of MTX. Braun et al (8) recently reported the results of a clinical trial showing that MTX is more effective when given subcutaneously instead of orally. With this mode of application, the unpredictability of resorption can be avoided. My coworkers and I (9,10) have reported that MTX accumulates in inflamed joints if it is covalently Christoph Fiehn, MD: Center for Rheumatic Diseases, Baden-Baden, Germany. Dr. Fiehn has received consulting fees, speaking fees, and/or honoraria from Medac (less than $10,000). Address correspondence and reprint requests to Christoph Fiehn, MD, Center for Rheumatic Diseases, Baden-Baden, Rotenbachtalstrasse 5, D-76530 Baden-Baden, Germany. E-mail: c.fiehn@rheumazentrum-baden.de. Submitted for publication August 26, 2008; accepted in revised form October 3, 2008. Arthritis & Rheumatism

Use of some plant extracts for protection of liver cells from toxic effects of Methotrexate

The aim of this study was to detect the efficacy of some plant extracts in protection of liver cells from toxic effects of anti-cancer drug Methotrexate by using metabolizing enzymes glutamic oxaloacetic transaminase (GOT), glutamic pyruric transinase (GPT), and alkaline phosphatase (ALP), as parameters. Aquatic and alcohclic extracts were prepared from the Seeds of Nigella sativa, (Citrus aurntifolia) and cardamom (Elptteria cardamam ), for each extract, four gradually increased doses were evaluated for their toxicity. Dose with no effect was selected for further investigation. Drug-plant extract interaction was conducted using white mice musculus Balb/c strain. The animals were divided into three groups: negative controls (buffer), positive control (drug) and interaction group (drug &amp; plant extract). The results showed that the dose 200 mg/kg, bw of N. sativa seeds extract and citrus fruit extract, as well as 50 mg/kg of cardamom extract induced non significant reduction in activity of GOT, GPT and ALP. However, MTX at dose of 3.25 mg/kg caused a significant (P&lt; 0.01) reduction in those enzymes. Interaction between the selected doses of drug and plant extract, before and after drug treatments, was conducted. Significant reduction of drug effects on GPT, GOT and ALP was noticed when N. sativa and cardamom extracts were given for 7 days before drug treatments. Citrus fruit and aquatic extract of cardamom were negative in such experiment. Patient on treatment with MTX should be checked for liver function test and these plants might protect him.

Role of liver function tests in detecting methotrexate-induced liver damage in sarcoidosis.

Methotrexate has become a standard second-line agent for the treatment of sarcoidosis. Because sarcoidosis has a high frequency of liver involvement, we routinely perform a liver biopsy after each cumulative gram of methotrexate therapy in patients with sarcoidosis in whom we plan to continue therapy. Following a previously described protocol for methotrexate therapy, we have performed 100 liver biopsies on 68 patients with chronic sarcoidosis at our institution. On the basis of the liver biopsy results, we identified the following 4 groups: sarcoidosis (47 cases), toxic effects of methotrexate (14 cases), hepatitis C (2 cases), and normal liver tissue (37 cases). We found no difference among the groups in terms of age, weight at time of biopsy, the number of patients receiving corticosteroids at the time of biopsy, cumulative dose of methotrexate, race, or sex. The 14 cases of toxic reactions to methotrexate included 5 patients who had undergone 1 or more previous liver biopsies in which the results did not show toxic effects. We found a significant difference between groups for levels of alkaline phosphatase and asparate aminotransferase at the time of starting (or restarting) methotrexate therapy (analysis of variance, P<.05). This finding was also true for the liver function tests performed at the time of the biopsy (analysis of variance, P<.05). The highest values were for those whose biopsy findings showed sarcoidosis. Toxic reactions to methotrexate eventually occurred in more than 10% of patients with sarcoidosis treated for more than 2 years with methotrexate. Because of hepatic involvement owing to sarcoidosis, results of serial liver function tests were not useful in determining which patients would have this reaction to methotrexate.

Potential transmission of the cytogenetic toxic effects of methotrexate in the male germline cells of Swiss mice

On testing the cytogenetic toxic effects of methotrexate, a widely prescribed antineoplastic drug, in the male germline cells of Swiss mice, it was found highly clastogenic to the spermatogonial cells at 24-h post-treatment after a single intraperitoneal exposure. The occurrence of significant percentages of aberrant primary spermatocytes with atypical bivalents at week 4 post-treatment and a little higher percentages of sperm with abnormal morphology at week 8 post-treatment indicated the potential transmission of the induced cytogenetic toxic effects of methotrexate from spermatogonia to sperm in the male germline cells of Swiss mice.

Survival gene transfer into leukemia specific donor lymphocytes for chemoprotection post allogeneic marrow transplantation

ALL is the least responsive of all leukemias to immunotherapy with DLI after allogeneic stem cell transplantation. The absence of the GVL effect is probably due to 1) poor immunogenicity of ALL cells and 2) a high proliferative capacity of the leukemia, resulting in the need for frequent chemotherapy to control the leukemic clone which has the deleterious effect of eradicating any expanding ALL-specific cytotoxic T-lymphocytes (CTLs). We have shown that the immunogenicity of ALL cells can be increased by upregulating essential (co)stimulatory molecules (B7.1&2, CD40, ICAM-1, LFA-3) using CD40 ligand. CD40 ligated ALL cells are highly efficient in inducing allogeneic T-cells to proliferate compared to untreated ALL cells. CD40ligated ALL cells can also be used to generate ALL-specific CTLs from the donor. The CTLs are protected from the toxic effects of methotrexate (MTX) or trimetrexate (TMTX) chemotherapy by introducing a drug resistant variant of dihydrofolate reductase (L22YDHFR). Gene modified ALL-specific CTLs will allow for the administration of antifolate chemotherapy which will control the leukemia and reduce the leukemia burden while the ALL-specific CTLs will be protected from the chemotherapy, expand, and eradicate the leukemia. Transduction efficiency of T-cells was measured by flow cytometry after gene modification with a replication incompetent lentivirus-based vector encoding Green Fluorescent Protein & L22YDHFR. After TMTX selection the transduction efficiency was 50% compared to 13% of T-cells not exposed to TMTX. We are currently investigating if L22YDHFR transduction significantly alters the function of antigen specific CTLs.

2,4‐Diamino‐7‐hydroxy‐pteridines, a new class of catabolites of methotrexate

Methotrexate remains a commonly used drug in the chemotherapy of various malignancies. The known catabolites are 7-hydroxy-methotrexate, formed in the liver, and diamino-methyl-pteroic acid formed in the gut. We report for the first time evidence that 2,4-diamino-7-hydroxy-pteridine derivatives are present in the biological fluids of patients on high-dose methotrexate protocols. So far, two major derivatives have been identified as 2,4-diamino-6-hydroxymethyl-7-hydroxy-pteridine and 2,4-diamino-6-methyl-7-hydroxy-pteridine. In regard to the actual knowledge of the catabolism of pteridines, these compounds are presumably formed by intestinal bacteria during enterohepatic circulation of the drug. Their slow clearance from the body raises the question of possible interference of these compounds on pteridine-dependent enzymes, which might explain in part some of the toxic effects of methotrexate.

Acute fatal myeloencephalopathy after combined intrathecal chemotherapy in a child with acute lymphoblastic leukemia

We report a case of a fatal toxic encephalomyelopathy in a 12-year-old girl due to prophylactic intrathecal injection of methotrexate and cytosine arabinoside, with a characteristic progressive symptomatology leading to death after 28 days. The location and type of neuropathological changes support the hypothesis of a direct toxic effect of methotrexate and/or cytosine arabinoside on structures directly exposed to the cerebrospinal fluid.