Folate Analogue Research Articles

Safety of Same-Day Vitamin B12 Supplementation in Patients Receiving Pemetrexed for the Treatment of Non–Small-Cell Lung Cancer or Pleural Mesothelioma: A Retrospective Analysis

BackgroundPemetrexed is a folate analog inhibitor for the treatment of non–small-cell lung cancer (NSCLC) and malignant pleural mesothelioma. Folic acid and vitamin B12 supplementation before initiating pemetrexed is necessary because of high rates of cytopenias without supplementation. However, the timing of supplementation has not been thoroughly investigated. Patients and MethodsThis was a single-center, retrospective study investigating patients receiving pemetrexed from January 1, 2012, to June 30, 2015, who received same-day vitamin B12 supplementation versus ≥ 1 day before pemetrexed. The objective was to evaluate safety outcomes in patients who received vitamin B12 on the same day as pemetrexed (group A) versus vitamin B12 ≥ 1 day (group B) before pemetrexed. ResultsTwo hundred eighty-one patients met the inclusion criteria: 137 patients in group A (same-day administration of vitamin B12) and 144 patients in group B (median time of vitamin B12 administration before pemetrexed, 7 days; range, 1-42 days). Mean changes in hematologic indices from cycle (C) 1 to C2 or C2 to C3 did not differ significantly between groups. There were no significant differences in clinical events between C1 and C2 or C2 and C3 requiring supportive care. There was a significant difference noted in treatment delay in C3 [28/114 (24.6%) group A vs. 14/118 (11.9%) group B, P = .0164]. In group A, significant predictors of delay in C3 were baseline hemoglobin (mean 13.3 g/dL vs. 12.4 g/dL, P = .0137) and ANC (mean 6 × 109/L vs. 5 × 109/L, P = .0003). ConclusionSame-day vitamin B12 and pemetrexed administration is a safe practice in NSCLC and malignant pleural mesothelioma patients.

Electrochemistry Study of Antineoplastic Raltitrexed Oxidation Mechanism and its Interaction with DNA

AbstractRaltitrexede (RTX) is a folate analogue that belongs to the antimetabolites family and has antineoplastic activity correlated to inhibition of the thymidylate synthase (TS) enzyme. This study addresses the electrochemical characterization of RTX at a glassy carbon electrode, on a wide interval of pH, using voltammetric techniques. The interaction between RTX and double helix DNA (dsDNA) in physiological medium is also addressed, using a DNA‐electrochemical biosensor. A mechanism for the electrochemical oxidation of RTX is proposed. RTX is irreversibly electroxidized under a predominantly diffusion controlled and pH‐dependent process. The oxidation process in acid and physiological media occurs in two consecutive steps. The first oxidation step is pH‐independent and associated with the transfer of one electron of the nitro group at N10 position by releasing a methyl cation. The second oxidation step is pH‐dependent and occurs with the transfer of one electron and one proton from the carbon at C9 position, followed by a direct attack by a water molecule leading to an irreversible dissociation of an oxidation product of RTX. A strong interaction between RTX and dsDNA was revealed by using a multilayer dsDNA electrochemical biosensor. The experiments demonstrated that RTX interacts and binds to the dsDNA helix by different interaction modes, suggesting an intercalation in between the DNA base pairs leading to defects in its secondary structure.

Dihydrofolate reductase and membrane translocation: evolution of a classic experiment: Classic landmark papers, irrespective of their age, can teach students how best science is practiced and inspire new experiments.

Any PhD student or postdoc, who scrambles to write and submit a publication before getting scooped, can testify that science is a fast‐moving endeavor. Given our limited time and the ever‐increasing pace with which scientific studies are published, few students and postdocs—and PIs as a matter of fact—have time to keep up with the literature. “Reading” a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. Notwithstanding these constraints, I would argue that the scientific literature is a treasure trove of information and ideas that go beyond contemporary papers to include classical publications. However, it can be difficult to motivate students and postdocs to read old landmark publications that reported groundbreaking discoveries and opened up new avenues of research. Many think that these papers are of merely historic interest and have little to contribute to scientists expected to use cutting‐edge methods to produce high‐impact publications. > Reading a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. However, classic papers still have a lot to offer. For once, they withstood the test of the time, as the reported results have shown to be correct and reproducible. This is unfortunately not the case for many high‐impact publications today, which, owing to the hype of selling it to the highest impact factor journal, just tell a cool story that in reality may be much less clear‐cut than reported. Furthermore, classic scientific publications often impress by a conceptual clarity and in many cases simplicity and thereby offer valuable lessons of how best science should be practiced. These are qualities I miss in many of today's papers that contain exceedingly large data sets …

PD48 OTL38, A Tumour-Specific Agent In Surgery For Ovarian Cancer

Introduction:Ovarian cancer (OC) has the highest mortality rate of all gynecologic malignancies. Completeness of cytoreductive surgery is a key prognostic factor for survival. To differentiate clearly between malignant and healthy tissue is essential for achieving complete cytoreduction. Using current approaches, this differentiation is difficult and can lead to incomplete tumour removal. OTL38 is a folate analogue conjugated to a near-infrared fluorescent dye which has high specificity and affinity for folate receptor alpha (FRα) expressed in OC. OTL38 together with a specific imaging system can help the surgeon to visualize diseased tissue. The objective of this work is to know the effectiveness and safety of OTL38 in of OC surgery.Methods:Early assessment of OTL38 identified through the early-awareness and alert-system, “SINTESIS-new technologies”, of AETS-ISCIII. The searched databases were: PubMed, WOS, Tripdatabase, Dynamed, Cochrane Library and ICTRP. Clinical studies using the OTL38 in cytoreductive surgery in OC published until September 2017 were reviewed.Results:Only one publication, supported by industry, was retrieved. The study assesses the pharmacokinetics and tolerability of OLT38 in 30 healthy people randomized into 4 groups with different doses and a control group. The study also analyses the percentage of cytoreduction in 12 OC patients. Infusion of 0.025, 0.05, and 0.1 mg/kg OTL38 doses was associated with mild adverse events which did not require intervention. The 0.2 mg/kg dose was associated to adverse events of moderate severity. In OC patients, 0.0125 mg/kg dose was considered the optimal dose with mild adverse events. OTL38 accumulated in FRα-positive tumours and metastases allowed the surgeon to resect an additional 29 percent of malignant lesions which were not detected using standard inspection and palpation methods.Conclusions:The OTL38 is an emergent health technology, which could help in cytoreductive surgery for ovarian cancer. However, the evidence is scarce and it would be necessary to continue further studies.

Folate receptor overexpression can be visualized in real time during pituitary adenoma endoscopic transsphenoidal surgery with near-infrared imaging.

OBJECTIVE Pituitary adenomas account for approximately 10% of intracranial tumors and have an estimated prevalence of 15%-20% in the general US population. Resection is the primary treatment for pituitary adenomas, and the transsphenoidal approach remains the most common. The greatest challenge with pituitary adenomas is that 20% of patients develop tumor recurrence. Current approaches to reduce recurrence, such as intraoperative MRI, are costly, associated with high false-positive rates, and not recommended. Pituitary adenomas are known to overexpress folate receptor alpha (FRα), and it was hypothesized that OTL38, a folate analog conjugated to a near-infrared (NIR) fluorescent dye, could provide real-time intraoperative visual contrast of the tumor versus the surrounding nonneoplastic tissues. The preliminary results of this novel clinical trial are presented. METHODS Nineteen adult patients who presented with pituitary adenoma were enrolled. Patients were infused with OTL38 2-4 hours prior to surgery. A 4-mm endoscope with both visible and NIR light capabilities was used to visualize the pituitary adenoma and its margins in real time during surgery. The signal-to-background ratio (SBR) was recorded for each tumor and surrounding tissues at various endoscope-to-sella distances. Immunohistochemical analysis was performed to assess the FRα expression levels in all specimens and classify patients as having either high or low FRα expression. RESULTS Data from 15 patients (4 with null cell adenomas, 1 clinically silent gonadotroph, 1 totally silent somatotroph, 5 with a corticotroph, 3 with somatotrophs, and 1 somatocorticotroph) were analyzed in this preliminary analysis. Four patients were excluded for technical considerations. Intraoperative NIR imaging delineated the main tumors in all 15 patients with an average SBR of 1.9 ± 0.70. The FRα expression level of the adenomas and endoscope-to-sella distance had statistically significant impacts on the fluorescent SBRs. Additional considerations included adenoma functional status and time from OTL38 injection. SBRs were 3.0 ± 0.29 for tumors with high FRα expression (n = 3) and 1.6 ± 0.43 for tumors with low FRα expression (n = 12; p < 0.05). In 3 patients with immunohistochemistry-confirmed FRα overexpression (2 patients with null cell adenoma and 1 patient with clinically silent gonadotroph), intraoperative NIR imaging demonstrated perfect classification of the tumor margins with 100% sensitivity and 100% specificity. In addition, for these 3 patients, intraoperative residual fluorescence predicted postoperative MRI results with perfect concordance. CONCLUSIONS Pituitary adenomas and their margins can be intraoperatively visualized with the preoperative injection of OTL38, a folate analog conjugated to NIR dye. Tumor-to-background contrast is most pronounced in adenomas that overexpress FRα. Intraoperative SBR at the appropriate endoscope-to-sella distance can predict adenoma FRα expression status in real time. This work suggests that for adenomas with high FRα expression, it may be possible to identify margins and to predict postoperative MRI findings.

Methotrexate and epirubicin conjugates as potential antitumor drugs.

The use of hybrid molecules has become one of the most significant approaches in new cytotoxic drug design. This study describes synthesis and characterization of conjugates consisting of two well-known and characterized chemotherapeutic agents: methotrexate (MTX) and epirubicin (EPR). The synthesized conjugates combine two significant anticancer strategies: combinatory therapy and targeted therapy. These two drugs were chosen because they have different mechanisms of action, which can increase the anticancer effect of the obtained conjugates. MTX, which is a folic acid analog, has high cytotoxic properties and can serve as a targeting moiety that can reach folate receptors (FRs) overexpresing tumor cells. Combination of nonselective drugs such as EPR with MTX can increase the selectivity of the obtained conjugates, while maintaining the high cytotoxic properties. Conjugates were purified by RP-HPLC and the structure was investigated by MS and MS/MS methods. The effect of the conjugates on proliferation of LoVo, LoVo/Dx, MCF-7 and MV-4-11 human cancer cell lines was determined by SRB or MTT assay. The conjugation reaction results in the formation of monosubstituted (α, γ) and disubstituted MTX derivatives. In vitro proliferation data demonstrate that the conjugates synthesized in our study show lower cytotoxic properties than both chemotherapeutics used alone. Epirubicin cytotoxicity was not observed in obtained conjugates. Effective drugs release after internalization needs further investigation.

Development of a lateral flow dipstick immunoassay for evaluation of folate levels in maize.

Folates (vitamin B9) are essential for all organisms as cofactors for one-carbon metabolism. However, measurement of folates is technically complicated and time-consuming. In this study, we developed a dipstick immunoassay using a folate-specific monoclonal antibody (mAb), allowing rapid and low-cost detection of folates. The indicator range of the dipstick for 5-formylterahydrofolate (5-CHO-THF), 5-methyltetrahydrofolate (5-CH3-THF) and their polyglutamyl forms was 100-200ngmL-1; moreover, no cross-reactivity was observed with tetrahydrofolate (THF) or 5,10-methenyltetrahydrofolate (5,10-CH=THF) at 500ngmL-1, or with the folate precursors pterin-6-COOH, p-aminobenzoate (pABA), and L-glutamate, or with the folate analogues methotrexate and 10-formyltetrahydrofolate (10-CHO-THF) at up to 1000ngmL-1. The dipstick immunoassay was tested in maize seeds; the results classified the seeds into those with low, moderate, and high levels of folates, and were in agreement with those of liquid chromatography-mass spectrometry. Thus, we conclude that the dipstick assay will provide a versatile tool to facilitate large-scale screening of maize rich in folates. Graphical Abstract The dipstick based immunoassay for analyzing folate level in maize.

Abstract 5491: Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors

Abstract The majority of folate uptake into tissues and tumors involves facilitated carriers, the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). PCFT is highly expressed in a wide range of human tumor cell lines and primary tumor specimens from a variety of lineages, with modest expression in most normal tissues. PCFT transports optimally at pHs characterizing the tumor microenvironment. We discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine folate analogs with substantial selectivity for PCFT over RFC, resulting in potent antitumor efficacy in vitro and in vivo. However, in some cases PCFT levels varied substantially. Studies were performed to characterize the basis for these dramatic differences in PCFT gene expression between HepG2 (elevated) and HT1080 (negligible) tumor cells. Low levels of PCFT gene expression in HT1080 cells were not likely due to methylation of CpG islands in the PCFT promoter since 5-aza-2’-deoxycytidine treatment did not restore PCFT gene expression. A PCFT promoter reporter construct -2005/+96 in pGL3 (transcription starts at +1) was generated and showed high levels of activity in HeLa cells by luciferase reporter assays. For HepG2 and HT1080 cells, wide variations in PCFT transcript levels by real-time RT-PCR were paralleled by reporter gene activities with the -2005/+96 construct, suggesting that these were primarily due to PCFT transcriptional regulation. Progressive deletion analysis of the -2005/+96 construct and reporter assays in HepG2 and HT1080 cells localized the minimal promoter region to between -50 and +96. Additional deletion constructs (-35/+96, -20/+96, -15/+96 and -10/+96) were generated and studied. Whereas the -35/+96, -20/+96, -15/+96 promoter constructs retained ~50% activity for HepG2 cells and ~10-15% activity for HT1080 cells relative to the -50/+96 construct, in both cases the -10/+96 construct was essentially inactive. Two critical cis elements were identified in the PCFT minimal promoter by deletion analysis located between positions -50 and -35, and -15 and -10. Bioinformatics analysis localized putative cis elements for KLF-15 (-50 to -35) and NRF1 (-15 to -10) binding. The functional importance of these elements was confirmed by mutations of core consensus sequences in the -50/+96 construct and reporter gene assays. Binding of NRF1 and KLF15 to the endogenous PCFT promoter in HepG2 and HT1080 cells was confirmed by ChIP assays which paralleled patterns of differential PCFT gene expression. Collectively, our results suggest that NRF1 and KLF15 contribute to dramatic variations in PCFT gene expression between tumor cells. Better understanding the key determinants of PCFT transcriptional control may lead to strategies for modulating PCFT levels in combination with PCFT-targeted 6-pyrrolo[2,3-d]pyrimidine folate analogs for treating solid tumors. Citation Format: Zhanjun Hou, Carrie O'Connor, Steve Orr, Larry H. Matherly. Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2017-5491

Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase

The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.

Silver nanoparticle-embedded graphene oxide-methotrexate for targeted cancer treatment

Combination therapies are widely investigated cancer treatment modalities. Carbon based systems such as graphene oxide (GO), plasmonic nanoparticles such as silver nanoparticles (AgNPs), and the folate analog, methotrexate (MTX), have been separately studied for their potential anticancer effects. In this study, we combined these systems to develop AgNPs-embedded GO with conjugated MTX (MTX-GO/AgNPs) and studied their folate receptor-targeted anticancer effects. Results revealed successful formation of AgNPs on GO along with MTX conjugation as suggested by UV/visible, TEM, AFM, FTIR, and XRD analysis. Folate receptor-positive MCF-7 cells were more prone to cytotoxic effects of MTX-GO/AgNPs compared to folate receptor-negative HepG2 cells. Folic acid analog MTX interacts with folate receptors expressed in MCF-7 cells, improving cellular uptake and subsequent anticancer effects of the system. Importantly, AgNPs enhanced the total ROS production within the treated cells leading to improve cellular apoptosis, as evidenced by western blot. Moreover, near infrared (NIR)-induced photothermal effects of GO improved the anticancer activity of the system. Therefore, the combinational therapy system MTX-GO/AgNPs can be potentially applied for effective folate receptor-targeted treatment of cancers.

CHOP in Combination with Pralatrexate, a Novel Folate Analogue Metabolic Inhibitor in Patients with Previously Untreated Peripheral T-Cell Lymphoma (PTCL): Interim Results of the Phase 1 Trial

Background: Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of mature T-cell and natural-killer-cell aggressive non-Hodgkin lymphomas associated with poor prognosis with current therapies. Numerous studies have reported poor survival for PTCL patients with a median Overall Survival (OS) shorter than 2 years and 5-year survival rates of <30%. Traditionally, patients with PTCL have been treated with a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-like first-line chemotherapeutic regimen. Unfortunately, the prognosis remains poor with most patients relapsing within 2 years. Thus, improved front line treatment strategies are needed.Pralatrexate (Folotyn) is a folate analogue metabolic inhibitor that was the first drug approved in the US for the treatment of patients with relapsed or refractory PTCL (R/R PTCL). Approval was based on results from the pivotal Phase 2 PROPEL study (O'Connor et al, JCO, 2011) of pralatrexate in R/R PTCL, in which pralatrexate monotherapy demonstrated a durable clinical benefit (objective response rate of 29%). Pralatrexate is administered intravenously (IV) at a dose of 30 mg/m2 weekly for 6 weeks of a 7-week treatment cycle. Since pralatrexate and each of the components of the CHOP regimen target different aspects of tumor cell growth and proliferation, there is a potential for synergistic anti-tumor effect, and limited additive toxicities.Objectives: The primary endpoint of the study is the determination of the MTD of pralatrexate in combination with CHOP (Fol-CHOP) in previously untreated PTCL patients. Secondary endpoints included safety, tolerability and ORR (complete response [CR] + partial response [PR]) and pharmacokinetics.Methods: In this study, patients with PTCL received CHOP in combination with pralatrexate, repeated every 3-weeks for up to 6 cycles. Part 1 of the study was to determine the Maximum Tolerated Dose (MTD) of the Fol-CHOP combination. Once the MTD was determined, an additional 10 patients were treated in the Expansion Phase (Part 2). The cohort schema followed a traditional "3+3" design. Pralatrexate was to be administered as 10, 15, 20, 25, or 30 mg/m2 IV push over 3 to 5 minutes on Days 1 and 8 of each cycle. The starting cohort was CHOP + 10 mg/m2 of pralatrexate.Patients received prophylaxis with acyclovir and sulfamethoxazole/trimethoprim during the study and primary prophylaxis with growth factor (filgrastim or pegfilgrastim) support. Dose-limiting toxicities (DLT) were considered during the 1st cycle and included: Severe infections, study treatment-related non-hematological toxicity Grade 3-4, platelet count < 25 X 109/L at any time or ANC < 0.5 X 109/L lasting more than 7 days despite G-CSF administration.Results: A total of 12 patients have been enrolled in Part 1 of the study at the time of abstract submission. Nine patients have completed Cycle 1 and no DLT was reported. Among these nine patients, three patients each were treated at CHOP + 10 mg/m2, 15 mg/m2 and 20 mg/m2 of pralatrexate. One patient (11%) treated with CHOP + 10 mg/m2 had one serious adverse event (SAE) of febrile neutropenia that was deemed unrelated to any study drug. Only four Grade 3 AEs (anemia, constipation, mucosal inflammation, and nausea) were reported, in one patient (11%). The enrollment into the study continues.Conclusions: These interim results demonstrate that the combination of pralatrexate with CHOP (Fol-CHOP) is well tolerated, with all components of CHOP as well as pralatrexate being given at close to or at standard doses. The rates of AEs are comparable to those typically reported with CHOP alone. The study is ongoing to confirm the MTD and to assess preliminary efficacy of the Fol-CHOP combination. Updated results will be presented at the meeting. DisclosuresOki:Novartis: Research Funding. Barta:Celgene, Merck, Seattle Genetics: Research Funding; Janssen: Honoraria, Speakers Bureau. Sharma:Spectrum Pharmaceuticals: Employment. Song:Spectrum Pharmaceuticals: Employment. Mourya:Spectrum Pharmaceuticals: Employment.

Self-Delivery Nanoparticles of Amphiphilic Methotrexate-Gemcitabine Prodrug for Synergistic Combination Chemotherapy via Effect of Deoxyribonucleotide Pools.

The distinct and complementary biochemical mechanisms of folic acid analog methotrexate (MTX) and cytidine analog gemcitabine (GEM) make their synergistic combination effective. Unfortunately, such a combination faces severe pharmacokinetic problems and several transportation barriers. To overcome these problems, a new strategy of amphiphilic small molecule prodrug (ASMP) is developed to improve their synergistic combination effect. The ASMP was prepared by the amidation of the hydrophilic GEM with the hydrophobic MTX at a fixed ratio. Owing to its inherent amphiphilicity, the MTX-GEM ASMP self-assembled into stable nanoparticles (ASMP-NPs) with high drug loading capacity (100%), in which the MTX and GEM could self-deliver without any carriers and release synchronously in cancer cells. In vitro studies showed that the MTX-GEM ASMP-NPs could greatly improve the synergistic combination effects by the reason of arresting more S phase of the cell cycle and reducing levels of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), and deoxycytidine triphosphate (dCTP). The stronger synergistic effects caused the higher cell cytotoxicity and apoptotic ratio, and circumvented the multidrug resistance (MDR) of tumor cells. Additionally, MTX-GEM ASMP-NPs could achieve the same anticancer effect with the greatly reduced dosage compared with the free drugs according to the dose-reduction index (DRI) values of MTX and GEM in MTX-GEM ASMP-NPs, which may be beneficial for reducing the side effects.

Hepatoxicity in a Patient with Acute Lymphoblastic Leukemia Receiving High-Dose Methotrexate

Introduction: Methotrexate (MTX) is a folic acid analogue that inhibits purine and pyrimidine synthesis. The dosing regimen varies by indication; high, short term doses are used in the treatment of cancer and chronic, lower doses for autoimmune conditions. The pattern of liver toxicity is dose dependent. We present a case of high dose methotrexate induced acute liver injury. Case: A 31 year old female with a history of pre-B cell acute lymphocytic leukemia (ALL), and treated with one cycle of hyper-CVAD (Cyclophosphamide, Vincristine, Doxorubicin and Dexamethasone) was admitted for part B of her chemotherapy (MTX and cytarabine). Upon admission, she received 1000 mg/m2 of intravenous MTX. Leucovorin was administered and urine alkalinization performed per protocol. However, the patient's serum MTX levels remained elevated up to 96 hours after administration. Two days after MTX treatment her liver function tests (LFTs) became elevated from previously normal levels, with ALT 1608, AST 752, Alkaline Phosphatase 178, and Total Bilirubin 2.8. The LFTs peaked five days after the initial dose. The patient did not have encephalopathy, coagulopathy or jaundice. After other etiologies of acute liver injury were ruled out, the elevation of LFTs was determined to be related to MTX. LFTs began down trending 6 days after MTX dose. Approximately one month after administration of MTX the patient's LFTs normalized. Discussion: Methotrexate is an important part of ALL chemotherapy. With high dose intravenous MTX, serum ALT levels can rise to 10 to 20 times the upper limit of normal within 12 to 48 hours, but levels then rapidly return to normal with only rare instances of clinical liver injury. Gene polymorphisms play important role in MTX toxicity in ALL patients. It is important to assess for underlying liver disease in patients with excessive alcohol use, chronic viral hepatitis, elevations in serum enzymes, and in selected patients with diabetes and obesity before MTX therapy. Leucovorin is given in conjunction with high dose MTX to prevent death of non-malignant cells. However, this has not been proven to prevent hepatoxicity. Treatment of hepatotoxicity caused by MTX includes cessation of the drug and observation of LFTs. If LFTs do not normalize then liver biopsy is indicated to determine extent of damage. Liver injury can be minimized with MTX therapy by avoiding alcohol and hepatotoxic drugs, supplementing folic acid and closely observing LFTs.

Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC–MS

Pralatrexate (PTXT) is an antineoplastic folate analog and the chemical name is (2S)-2-[[4-[(1RS)-1-[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl] benzoyl] amino] pentanedioic acid. Degradation products of PTXT drug product (DP) under different forced degradation conditions have been studied using LC-PDA and LC–MS techniques. PTXT DP was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, and heat in accordance with ICH guidelines. The LC–MS compatible HPLC method was developed and stressed solutions were chromatographed on reversed phase HPLC. The degradation products were monitored at a wavelength of 242nm. Stress study revealed that PTXT was sensitive towards acid, alkali, peroxide, light and heat. The degradation impurities (I–IX) were identified and characterized using LC-PDA and mass spectral data.

Insights on methotrexate in psoriatic disease

The folic acid analogue methotrexate is used as an anti-neoplastic agent and treatment for inflammatory disorders including psoriasis, dermatomyositis, lupus erythematous, sarcoidosis, and systemic sclerosis. Despite the introduction of newer biologic agents, methotrexate remains a first-line systemic therapy for many patients with disorders of chronic inflammation. Here we briefly describe the current clinical evidence for methotrexate use in psoriatic disease, our current understanding of methotrexate's anti-inflammatory properties, and the future role of methotrexate in the treatment of immune mediated disorders.

Protective effect of Morinda citrifolia L. (fruit extract) on methotrexate-induced toxicities—hematological and biochemical studies

AbstractMethotrexate (MTX) has been widely used as an anticancer drug. It acts as a folic acid analog, inhibits purine and pyrimidine synthesis, which accounts for its efficacy in the therapy of cancer as well as for some of its toxicities. The present study is an attempt of modulating MTX-induced toxicities using aqueous extract of Morinda citrifolia L. (Noni) as a nutritional supplement. Hematological parameters such as RBC, WBC, and platelet count that decreased (p < 0.05) after methotrexate injection was found to have been restored in rats co-treated with Noni. Enhanced levels of lipid peroxides (p < 0.05) in animals administered with MTX showed significant revision after co-administration of Noni. Alterations of other biochemical constituents in blood like glucose, urea, uric acid, and cholesterol (p < 0.05) were also reversed to near normal levels in animals co-treated with Morinda citrifolia L. The study provides preliminary evidence that Morinda citrifolia L. extract can ameliorate MTX mediated si...

Reflections on Henry Kunkel outside the laboratory

The folic acid analogue methotrexate is used as an anti-neoplastic agent and treatment for inflammatory disorders including psoriasis, dermatomyositis, lupus erythematous, sarcoidosis, and systemic sclerosis. Despite the introduction of newer biologic agents, methotrexate remains a first-line systemic therapy for many patients with disorders of chronic inflammation. Here we briefly describe the current clinical evidence for methotrexate use in psoriatic disease, our current understanding of methotrexate's anti-inflammatory properties, and the future role of methotrexate in the treatment of immune mediated disorders.

Abstract 1365: Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC

Abstract Drugs usually bind to different targets in different conformations. As antitumor agents, antifolates first need to be selectively transported by tumor cells via the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) or folate receptors (FRs). The second step is the binding of antifolates with the intracellular targets. We are systematically developing novel folate analogs with selective membrane transport for FRs and PCFT over the ubiquitously expressed RFC. These 6-substituted pyrrolo[2,3-d]pyrimidine antifolates are principally inhibitors of de novo purine biosynthesis at the steps catalyzed by glycinamide ribonucleotide formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). We previously demonstrated that classical antifolates bind to FRα and GARFTase in different conformations. An amide in the bridge region of classical antifolates has two interchangeable lowest energy conformers, pseudo-cis and pseudo-trans. We reported that a novel antifolate (AGF238) with an amide in the bridge region binds to FRα in a pseudo-trans conformation and binds to GARFTase in a pseudo-cis conformation. As part of our continued efforts to pursue targeted antifolates as antitumor agents, we designed an expand series of amide-bridged classical antifolates with different energy barriers between the pseudo-cis and pseudo-trans conformers. Among these, AGF266 was exclusively transported by Chinese hamster ovary (CHO) cells engineered to express human FRα (IC50 = 0.42 nM) or FRβ (IC50 = 0.88 nM) over CHO expressing human RFC (IC50 &amp;gt; 1000 nM). AGF266 was also found to exhibit highly potent antitumor activity (IC50 = 0.79 nM in KB human tumor cells). AGF266 inhibited GARFTase as its principal cellular target, based on patterns of protection from growth inhibition by adenosine and AICA. Our in vitro findings of antitumor activity associated with FR selectivity suggest that further preclinical evaluation of AGF266 as a potential antitumor agent is warranted. Citation Format: Aleem Gangjee, Weiguo Xiang, Aamod Dehkne, Mike R. Wilson, Adrianne Wallace-Povrik, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1365.

Abstract 4725: Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets

Abstract RX-3117 (Fluorocyclopentenylcytosine; FCPEC) is an orally bioavailable novel cytidine analog which is currently being evaluated in a Phase I clinical study. Downregulation of DNA methyltransferase 1 (DNMT1) by RX-3117 was shown in various cell lines with different histological backgrounds. In the present study we determined the effect of RX-3117 on DNMT1 at the DNA, RNA, protein and enzyme activity, and reactivation of suppressed target genes, including p16INK4A, methylguanine methyltransferase (MGMT) and the proton coupled folate transporter (PCFT). In the moderately sensitive non-small cell lung cancer (NSCLC) cell lines such as A549 and SW1573, 5-50 μM RX-3117 downregulated DNMT1 protein expression by 5-20% after 24 hr exposure and &amp;gt;90% after 48 hr. DNMT1 mRNA was not affected after 24 hr exposure but was affected moderately after 48 hr. In the sensitive ovarian cancer cell line A2780, protein down regulation was already observed after 24 hr at 1 μM RX-3117. DNMT1 activity was inhibited by 1 μM RX-3117 by 32% which was similar to the percent inhibition with 5 μM of the reference compound 5-aza-2’-deoxycytidine (DAC, 31%). In A549 cells 5 μM RX-3117 decreased overall methylation of DNA (detected by an antibody against 5-methylcytosine) by 25% after 48 hr exposure, while 5 μM DAC only inhibited 9%. For several genes known to be affected by methylation, we evaluated their protein expression and activity. In A549 and SW1573 cells a 24 hr exposure to 5 μM RX-3117 increased the expression of the cell cycle protein p16INK4A and of the DNA repair enzyme MGMT. For PCFT we evaluated its functional activity in CCRF-CEM leukemic cells which have a highly methylated PCFT promoter and in CEM-MTX cells which are deficient for the reduced folate carrier (RFC). PCFT is a specific folate transporter responsible for uptake of folic acid and the folate analogs methotrexate (MTX) and pemetrexed. Incubation of both CEM and CEM-MTX cells with either 29.6 μM RX-3117 or DAC as a positive control markedly increased PCFT mediated transport of MTX. This was more pronounced in CEM-MTX cells, 10-11-fold increase for both RX-3117 and DAC, compared to a 4-fold increase in CEM cells. In conclusion, the down-regulation of DNMT1 by RX-3117 was accompanied by a decrease in DNMT1 protein expression and enzyme activity, resulting in up-regulation of proteins of MGMT, PCFT, and the tumor suppressor gene p16INK4A. These data underline DNMT1 inhibition as a novel mechanism of RX-3117. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Joris J. Julsing, Ahmed Hassan, Kees Smid, Ietje Kathmann, Young Lee, Deog J. Kim. Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4725.

Genotoxic Effect of Methotrexate on Bone Marrow Chromosomes and DNA of Male Albino Mice (Mus Musculus)

Aim of the work-Methotrexate (MTX), a structural analogue of folic acid, is an antineoplastic and antirheumatic agent which is used in a variety of clinical schedules and combination therapy regimens in man. Material and methods- Sixty mice of nearly the same age were randomly categorized into four groups (one control and three treated groups with different doses of methotrexate). Mice of the treated groups 1, 2 and 3 were intraperitoneally injected with a single dose of methotrexate (2.5, 5 or 10 mg/kg b. wt. respectively) at the first day of the experiment. All the control and the treated animals were sacrificed after 24, 48 or 72 hour by cervical dislocation post treatment. Results-Methotrexate treatment induced structural and numerical chromosomal aberrations in male mice bone marrow cells which were significantly increased (P< 0.001) by dose and time. Structural aberrations were chromosomal gap, fragment, break, centromeric attenuation, deletion, centric fusion, ring formation, end to end association and beaded chromosomes. Numerical aberration was polyploidy. Also, methotrexate treatment decreased the mitotic index in bone marrow cells of all the treated mice in comparison with the control group by increasing dose and time of treatment. Comet assay results indicated that treatment with methotrexate significantly increased (P< 0.001) DNA damage in the blood leukocytes in dose and time dependent manner. Conclusion- It can be concluded that methotrexate induced genetic damage on the chromosomes and DNA content of male albino mice even after single treatment with low doses.