Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis.

A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX) and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug strategy selected is based on ROS-labile 4-methylphenylboronic acid promoieties linked to the drugs via a carbamate linkage or a direct C-N bond. Activation under pathophysiological concentrations of H2O2 proved to be effective, and prodrug candidates were selected in agreement with relevant in vitro physicochemical and pharmacokinetic assays. Selected candidates showed moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the expected safer toxicity profile and increased therapeutic window compared to MTX and AMT while maintaining a comparable therapeutic efficacy, which is highly encouraging for future use in RA patients.

Phase I/II study of pralatrexate in Japanese patients with relapsed or refractory peripheral T-cell lymphoma.

Pralatrexate is a novel antifolate approved in the USA for the treatment of relapsed or refractory peripheral T-cell lymphoma. To assess its safety, efficacy, and pharmacokinetics in Japanese patients with this disease, we undertook a phase I/II study. Pralatrexate was given i.v. weekly for 6 weeks of a 7-week cycle. All patients received concurrent vitamin B12 and folic acid. In phase I, three patients received pralatrexate 30 mg/m2 and none experienced a dose-limiting toxicity. In phase II, we treated 22 additional patients with that dose. The median number of treatment cycles was 1 (range, 1-9). Nine of 20 evaluable patients (45%) achieved an objective response by central review, including two complete responses. All responses occurred within the first treatment cycle. At the time of data cut-off, median progression-free survival was 150 days. Median overall survival was not reached. In the total population, the most commonly reported adverse events included mucositis (88%), thrombocytopenia (68%), liver function test abnormality (64%), anemia (60%), and lymphopenia (56%). Grade 3/4 adverse events included lymphopenia (52%), thrombocytopenia (40%), leukopenia (28%), neutropenia (24%), anemia (20%), and mucositis (20%). The pharmacokinetic profile showed no drug accumulation with repeat dosing. These results indicate that pralatrexate is generally well tolerated and effective in Japanese patients with relapsed or refractory peripheral T-cell lymphoma. This trial was registered with ClinicalTrials.gov (NCT02013362).

Phase 1 study evaluating the safety and pharmacokinetics of pralatrexate in relapsed/refractory advanced solid tumors and lymphoma patients with mild, moderate, and severe renal impairment.

PURPOSE: Pralatrexate is a folate analogue indicated for the treatment of relapsed or refractory peripheral T-cell lymphoma. It has not been formally tested in patients with renal impairment. This study evaluated the pharmacokinetic (PK) profile of pralatrexate in patients with renal impairment and with relapsed/refractory advanced solid tumors and lymphoma. METHODS: This was an open-label, nonrandomized, phase 1 study. Eligible patients received pralatrexate administered as an IV push over 3-5 min once weekly for 6 weeks in 7-week cycles until progressive disease or intolerable toxicity. Four cohorts of 6 patients were planned for a total of 24 patients. Patients with normal renal function (Cohort A), mild (Cohort B), and moderate renal impairment (Cohort C) received 30 mg/m2 pralatrexate once weekly for 6 weeks in 7-week cycles, and patients with severe renal impairment (Cohort D) were to be administered 20 mg/m2 once weekly for 6 weeks. Plasma and urine samples were collected at pre-specified time points to determine the PK profile of pralatrexate in each treatment cohort. Patients were followed for safety and tolerability. RESULTS: A total of 29 patients were enrolled and 27 patients (14 male) received at least 1 dose of pralatrexate. Because of a qualifying toxicity in Cohort C, the starting dose for Cohort D was reduced to 15 mg/m2. Chronic renal impairment led to a decrease in renal clearance of the pralatrexate diastereomers, PDX-10a and PDX-10b, but systemic exposure to these diastereomers was not dramatically affected by renal impairment. Pralatrexate exposure in Cohort D (15 mg/m2) was similar to the exposure in other cohorts (30 mg/m2). No apparent difference in toxicity between the four treatment cohorts was observed, except for an increase in cytopenias in patients with severe renal impairment. CONCLUSION: Pralatrexate exposure, at a dose of 30 mg/m2, in patients with mild or moderate renal impairment was similar to the exposure in patients with normal renal function. For patients with severe renal impairment only, a pralatrexate dose of 15 mg/m2 is recommended.

Pralatrexate Monitoring Using a Commercially Available Methotrexate Assay to Avoid Potential Drug Interactions.

Pralatrexate (PDX) is a folate antagonist structurally similar to methotrexate (MTX). Unlike MTX, it is currently not known whether PDX exhibits delayed clearance and heightened toxicity in the setting of fluid overload. A specific serum assay for PDX is not commercially available. To our knowledge, we report the first case using an MTX serum assay as a surrogate for PDX concentrations to avoid a potential drug-drug interaction with pralatrexate. We describe a 76-year-old man with refractory cutaneous T-cell lymphoma who began therapy with weekly PDX 15 mg/m(2) intravenous infusions on days 1, 8, and 15 of a 28-day cycle. He subsequently developed mucositis, a moderate right-sided pleural effusion, and peripheral edema over the next 5 weeks. Aggressive diuresis with furosemide was initiated, which was then withheld the day before his next PDX dose to avoid a potential drug-drug interaction between PDX and furosemide. His baseline MTX/PDX concentration (measured prior to administration of the cycle 2, week 2 PDX dose) was less than 0.20 µmol/L (i.e., undetectable). After PDX administration, his 1-hour peak MTX/PDX concentration increased to 0.58 µmol/L. Aggressive diuresis was withheld until his MTX/PDX concentration was undetectable, 43.5 hours later. PDX is more potent than MTX and displays similar pharmacokinetic properties. PDX concentrations using the serum MTX assay reflect lower values than those reported from PDX-specific assays in clinical studies. Because PDX is approved by the U.S. Food and Drug Administration for the treatment of uncommon malignancies, it is unlikely that a specific assay will be commercially developed. We propose that the MTX serum assay has merit for use in determining when to reinstate possible interacting drug therapies such as loop diuretics.

The potential of pralatrexate as a treatment of peripheral T-cell lymphoma.

INTRODUCTION: Peripheral T-cell lymphomas (PTCLs) are a group of rare malignancies originating from clonal proliferation of mature, post-thymic T cells that represent 10 - 15% of all non-Hodgkin's lymphomas with poor prognosis and median survival of 1 - 3 years. The standard treatment for PTCL has not yet been identified. Many patients with PTCL are refractory to first-line therapy. The complete response rate ranges from 36 to 66% according to different PTCL subtypes. Furthermore, those who reached a complete or partial response often have a shorter progression-free survival. AREAS COVERED: This paper discusses the potential of pralatrexate , a methotrexate analogue, as a treatment of PTCL. The authors report on the efficacy and safety data of controlled studies and describe the end points of ongoing trials. Pralatrexate was the first drug to obtain FDA approval for the treatment of patients with relapsed or refractory PTCL. However, the European Medicines Agency has refused marketing authorization. EXPERT OPINION: None of the treatments commonly used today have given satisfactory results. Pralatrexate seems to be one of the most promising agents in the treatment of patients with PTCL. Future efforts should be focused on better understanding the molecular pathogenesis of PTCL and on specific trials for different PTCL subtypes using rational drug combinations that include pralatrexate.

The major facilitative folate transporters solute carrier 19A1 and solute carrier 46A1: biology and role in antifolate chemotherapy of cancer.

This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (Solute Carrier 19A1) and the proton-coupled folate transporter (PCFT) (Solute Carrier 46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of health disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer, such as methotrexate and pralatrexate, are transported by RFC, and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases.

The membrane transport and polyglutamation of pralatrexate: a new-generation dihydrofolate reductase inhibitor.

PURPOSE: To characterize, directly and for the first time, the membrane transport and metabolism of pralatrexate, a new-generation dihydrofolate reductase inhibitor approved for the treatment for peripheral T-cell lymphoma. EXPERIMENTAL DESIGN: [(3)H]pralatrexate transport was studied in unique HeLa cell lines that express either the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT). Metabolism to active polyglutamate derivatives was assessed by liquid chromatography. These properties were compared to those of methotrexate (MTX). RESULTS: The pralatrexate influx K t, mediated by RFC, the major route of folate/antifolate transport at systemic pH, was 0.52 µΜ, 1/10th the MTX influx K i. The electrochemical potential of pralatrexate within HeLa cells far exceeded the extracellular level and was greater than for MTX. In contrast, MTX transport mediated by PCFT, the mechanism of folate/antifolate absorption in the small intestine, exceeded that for pralatrexate. After a 6 h exposure of HeLa cells to 0.5 µM pralatrexate, 80 % of intracellular drug was its active polyglutamate forms, predominantly the tetraglutamate, and was suppressed when cells were loaded with natural folates. There was negligible formation of MTX polyglutamates. The difference in pralatrexate and MTX growth inhibition was far greater after transient exposures (375-fold) than continuous exposure (25-fold) to the drugs. CONCLUSIONS: Pralatrexate's enhanced activity relative to MTX is due to its much more rapid rate of transport and polyglutamation, the former less important when the carrier is saturated. The low affinity of pralatrexate for PCFT predicts a lower level of enterohepatic circulation and increased fecal excretion of the drug relative to MTX.

Pralatrexate : evaluation of clinical efficacy and toxicity in T-cell lymphoma.

INTRODUCTION: Pralatrexate is a novel antifolate agent that belongs to the class of 10-deazaaminopterins. Its clinical efficacy as a single agent in relapsed or refractory peripheral T-cell lymphoma (PTCL) has been established in randomized trials. Treatment with this agent is generally safe. AREAS COVERED: This paper discusses the pharmacokinetics and efficacy of pralatrexate in T-cell lymphoma in clinical trials. In addition, the authors highlight pralatrexate-associated adverse effects and safety concerns. EXPERT OPINION: Although established as a second-line therapy, pralatrexate offers a clinical benefit to less than one-third of patients with PTCL. In addition, toxicity of this agent can be significant, especially mucositis, immunosuppression and thrombocytopenia. Currently, the potential synergy between pralatrexate and other agents in T-cell lymphoma is being explored in a number of studies. These results will hopefully prove the validity of this approach, leading to improved quantity of life in these patients, with an acceptable comfort index.

Pralatrexate: treatment of T-cell non-Hodgkin's lymphoma.

Pralatrexate is a folate analogue metabolic inhibitor manufactured by Allos Therapeutics, Inc., a wholly-owned subsidiary of Spectrum Pharmaceuticals, Inc. In both preclinical and clinical studies, pralatrexate demonstrated activity in lymphoma. Pralatrexate was US FDA approved for the treatment of relapsed/refractory peripheral T-cell lymphoma in 2009. Approval was based on data from the PROPEL trial that demonstrated an overall response rate of 29% in a heavily pretreated patient population. The dose and schedule of pralatrexate is 30-mg/m(2) weekly for 6 weeks, given in 7-week cycles. Folate and vitamin B12 supplementation are required to minimize toxicity. The most common toxicities are mucositis, thrombocytopenia, nausea and fatigue.

Pralatrexate: a novel synthetic antifolate for relapsed or refractory peripheral T-cell lymphoma and other potential uses.

PURPOSE: The pharmacology, pharmacokinetics, clinical trials, adverse effects, dosage, and economic considerations of pralatrexate (PDX) are reviewed. SUMMARY: Peripheral T-cell lymphoma (PTCL) comprises approximately 15-20% of all aggressive lymphomas and 5-10% of all non-Hodgkin's lymphomas. Advanced PTCL is often refractory to traditional first-line chemotherapy regimens. PDX was developed as a synthetic folate analog antimetabolite that competitively inhibits dihydrofolate reductase (DHFR). This results in the depletion of thymidine, leading to interference with deoxyribonucleic acid synthesis and cancer cell death. PDX has a higher potency than methotrexate and edatrexate (EDX). The efficacy and safety of PDX have been demonstrated in the PROPEL trial, a prospective phase II trial in patients with relapsed or refractory PTCL. Patients with prior stem cell transplantation receiving PDX also had similar response rates (RRs). PDX was investigated on the treatment of relapsed or refractory cutaneous T-cell lymphoma, previously treated advanced non-small cell lung cancer and other solid malignancies. PDX has similar side effects to other DHFR inhibitors. The most common side effect of PDX is mucositis. The recommended dose of PDX is 30 mg/m(2) weekly once for 6 weeks in 7-week cycle until disease progresses or unacceptable toxicity for PTCL and may require dose reduction or discontinuation. Patients should be supplemented with oral folic acid and intramuscular vitamin B(12) injections. CONCLUSION: PDX provides clinical benefit to patients with relapsed or refractory PTCL with durable complete and partial responses in patients who had not responded to multiple prior treatment regimens.

Pralatrexate with vitamin supplementation in patients with previously treated, advanced non-small cell lung cancer: safety and efficacy in a phase 1 trial.

INTRODUCTION: Pralatrexate is an antifolate designed for preferential tumor cell uptake and accumulation and received accelerated Food and Drug Administration approval in relapsed/refractory peripheral T-cell lymphoma. Pralatrexate 135 to 150 mg/m(2) every 2 weeks without vitamin supplementation was active in non-small cell lung cancer (NSCLC) although mucositis was dose limiting. This phase 1 study evaluated the safety of higher pralatrexate doses with vitamin supplementation to minimize toxicities. METHODS: Patients with stage IIIB/IV NSCLC received pralatrexate 150 to 325 mg/m(2) every 2 weeks with folic acid and vitamin B12 supplementation. Outcomes measured included adverse events (AEs), pharmacokinetics, and radiologic response. RESULTS: Thirty-nine patients were treated for a median of two cycles (range 1-16+). Common treatment-related grade 3 and 4 AEs by dose (≤190 mg/m(2) and >190 mg/m(2)) included mucositis (33 and 40%) and fatigue (11 and 17%). Treatment-related serious AE (SAE) rates for doses ≤190 and >190 mg/m(2) were 0 and 20%, respectively. The response rate was 10% (95% confidence interval: 1-20%), including two patients with complete response (26+ and 32+ months) and two with partial response. Serum pralatrexate concentrations increased dose dependently up to 230 mg/m(2). CONCLUSIONS: Pralatrexate with vitamin supplementation was safely administered to patients with previously treated NSCLC, and durable responses were observed. The recommended starting dose for phase 2 is 190 mg/m(2). A similar safety profile was observed in patients treated at 230 mg/m(2), although a higher serious AE rate was evident. Mucositis remains the dose-limiting toxicity of pralatrexate, and this study failed to demonstrate that vitamin supplementation prevents mucositis and failed to identify clinical predictors of mucositis. Individualized dose-modification strategies and prospective mucositis management will be necessary in future trials.

Evaluation of the pharmacokinetics, preclinical and clinical efficacy of pralatrexate for the treatment of T-cell lymphoma.

INTRODUCTION: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of T-cell neoplasms. Most patients with PTCL have a poor outcome with conventional therapies and are not cured without stem-cell transplantation. Pralatrexate, a novel antifolate chemotherapeutic agent, was rationally designed to impede folate metabolism by inhibiting dihydrofolate reductase (DHFR) and to be more efficiently internalized into tumor cells. Pralatrexate is the first drug that is FDA approved for patients with relapsed and refractory PTCL. AREAS COVERED: Pralatrexate has been used as a single agent and in combination with other agents in clinical trials for non-Hodgkin's lymphoma and Hodgkin's disease as well as in solid tumors. This review will cover the development of pralatrexate, the pharmacokinetics of pralatrexate, preclinical findings with pralatrexate and clinical studies of pralatrexate in hematologic malignancies. EXPERT OPINION: Pralatrexate has significant activity in vitro, and in early Phase I/II trials, responses were noted in patients with aggressive T-cell lymphomas. The Pralatrexate in Patients with Relapsed or Refractory Peripheral T-Cell Lymphoma trial demonstrated the activity of pralatrexate across a spectrum of heavily pretreated patients with different aggressive T-cell lymphoma subtypes, and studies in cutaneous T-cell lymphoma have shown efficacy at different doses and schedules. The most frequent adverse events in these trials were mucositis, reversible thrombocytopenia and fatigue.

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate.

INTRODUCTION: Folate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin. METHODS: Using a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans. RESULTS: EC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems. CONCLUSIONS: EC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

Transport of aminopterin by human organic anion transporters hOAT1 and hOAT3: Comparison with methotrexate.

The transport of antifolate aminopterin by human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) was characterized using Xenopus laevis oocytes and was compared with that of methotrexate. Although hOAT1 and hOAT3 transported both aminopterin and methotrexate, uptake of methotrexate was greater in hOAT3-expressing oocytes than in hOAT1-expressing oocytes, and aminopterin was transported by hOAT1 more efficiently. The apparent 50% inhibitory concentration (IC(50)) of aminopterin for p-aminohippurate uptake by hOAT1 was lower than that of methotrexate (methotrexate: 998 microM, aminopterin: 160 microM). On the other hand, IC(50) values of these antifolates for estrone sulfate transport by hOAT3 were comparable (methotrexate: 61.5 microM, aminopterin: 59.2 microM). The Michaelis-Menten constant and maximum velocity of aminopterin transport by hOAT1 were calculated to be 226 microM and 72.5 pmol/ oocyte/2 hr, respectively. Probenecid and non-steroidal anti-inflammatory drugs strongly inhibited the transport. These findings show that both aminopterin and methotrexate are substrates of hOAT1 and hOAT3, and that there are differences between the antifolates in terms of their transport characteristics.

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species.

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.

A population pharmacokinetic and pharmacodynamic evaluation of pralatrexate in patients with relapsed or refractory non-Hodgkin's or Hodgkin's lymphoma.

In a pralatrexate phase I study, patients displayed a high incidence of mucositis of grades 3 and 4. Preliminary evaluations of the pharmacokinetics of the drug and its association with mucositis suggested that pralatrexate exposure (area under the concentration-time curve (AUC)) could be controlled with body size (e.g., weight or body surface area)-based dosing and that pretreatment with folic acid and vitamin B(12) might diminish the incidence and severity of mucositis. The study was amended, with revised dosing and vitamin B(12) administration. Data from 47 patients were evaluated using NONMEM. Weight and methylmalonic acid (MMA) level were predictive of pharmacokinetic (PK) variability. AUC and MMA level were positively correlated with the risk of developing mucositis. A lower AUC schedule with vitamin B(12) pretreatment may control mucositis without compromising efficacy. The covariates identified in this study are comparable with other antifolate analogs. The application of modeling was a critical step in the development of pralatrexate, yielding important suggestions for dose, scheduling, and pretreatment modifications.

Phase 2B trial of aminopterin in multiagent therapy for children with newly diagnosed acute lymphoblastic leukemia.

PURPOSE: Aminopterin offers advantages over the related antifolate, methotrexate, including greater potency, complete bioavailability, and more consistent accumulation and metabolism by patients' blasts. This current trial was done to document the toxicity of the aminopterin within a multiagent therapeutic regimen for children with newly diagnosed ALL. EXPERIMENTAL DESIGN: Patients at high risk of relapse were non-randomly assigned to therapy including oral aminopterin 4 mg/m(2), in two doses 12 h apart, in place of methotrexate 100 mg/m(2) in four divided doses. RESULTS: Thirty-two patients, 22 with pre-B ALL and ten with T-lineage ALL, have been treated with aminopterin, with median follow up of 40 months. Hematologic, mucosal and hepatic toxicity has been tolerable and reversible. There have been no toxic deaths among patients in remission. During weekly AMT therapy, higher mean neutrophil counts were observed among patients who were wild type for polymorphisms in methylene tetrahydrofolate reductase and methionine synthase reductase. CONCLUSIONS: Aminopterin can be safely incorporated in multiagent therapy for patients with ALL, in place of systemic methotrexate, without causing excessive toxicity. These results support a larger trial comparing the efficacy and toxicity of aminopterin and methotrexate in therapy for patients with ALL.

Pharmacodynamic properties of methotrexate and Aminotrexate during weekly therapy.

4-Amino-pteroyl-glutamic acid (Aminotrexate; AMT) has several advantages over the related antifolate methotrexate (MTX), including greater potency, complete oral bioavailability, and greater accumulation by leukemic blasts in vitro. We compared the pharmacodynamic properties of AMT (given orally at 4 mg/m2 in two divided doses per week) and MTX (100 mg/m2 in four divided doses per week) among children with acute lymphoblastic leukemia. We find AMT and MTX to have equivalent penetration into the bone marrow compartment of these patients, as indicated by the steady-state concentrations within mature red blood cells (RBCs). However, MTX concentrations in the cerebrospinal fluid after oral dosage are significantly greater than AMT. To confirm these clinical observations, mice were treated four weekly injections of AMT or MTX, at a 1:20 dosage ratio, and tissue antifolate content was then determined over the subsequent 22 days. We confirm the selective exclusion of AMT from the CNS compartment, while showing equivalent accumulation of AMT and MTX in the RBCs, liver, spleen, kidneys and testes. Finally, we demonstrate that AMT, MTX, and their predominant polyglutamate species are equipotent inhibitors of their target intracellular enzyme dihydrofolate reductase, emphasizing the critical nature of steady-state tissue accumulation in determining the relative cytotoxic potency of these two antifolates.

Phase II trial of oral aminopterin for adults and children with refractory acute leukemia.

PURPOSE: To determine the antileukemic activity of weekly oral aminopterin in patients with refractory acute leukemia; to describe the pharmacodynamic properties of aminopterin; and to contrast the intracellular metabolism of aminopterin and methotrexate by patients' blasts in vitro. EXPERIMENTAL DESIGN: Forty-six patients were enrolled in three strata: children with acute lymphoblastic leukemia (ALL), adults with ALL, and patients with acute myeloid leukemia (AML). Aminopterin was given weekly, in two doses of 2 mg/m(2), 12 hours apart. Limited sampling pharmacokinetic analysis was done during the first week of therapy. Accumulation of [(3)H]aminopterin and [(3)H]methotrexate by leukemic blasts was studied in vitro. RESULTS: Six of 22 children with ALL (27%; 95% confidence interval, 8-47%) had clinically significant responses. None of those with AML and only two of 11 adults with ALL had responses meeting protocol definitions, although peripheral blast counts tended to decrease with therapy in all groups. Mucosal toxicity was minimal, even with limited use of leucovorin rescue. Complete bioavailability of aminopterin was confirmed, with a mean area under the curve of 0.52 +/- 0.03 micromol hour/L after oral dosing. No relationship between aminopterin pharmacokinetics and response was seen. In vitro, aminopterin showed more consistent metabolism by leukemic blasts to polyglutamates than methotrexate. Lineage-specific differences in the pattern of intracellular antifolylpolyglutamates were observed. CONCLUSIONS: Weekly oral aminopterin has significant activity among children with refractory ALL. With greater cellular accumulation and metabolism, more reliable bioavailability than methotrexate, and tolerable toxicity at this dose and schedule, aminopterin deserves further study as a potent alternative to methotrexate.

Final results of a phase I and pharmacokinetic study of gamma-methylene-10-deazaaminopterin (MDAM) administered intravenously daily for five consecutive days in patients with solid tumors.

PURPOSE: To determine the maximum tolerated dose (MTD) of gamma-methylene-10-deazaaminopterin (MDAM), a unique antifolate structurally similar to methotrexate (MTX), in the treatment of patients with solid tumors and to characterize toxicity and pharmacokinetic profiles of MDAM administered intravenously for five consecutive days repeated every 21 days. METHODS: A group of 18 patients with treatment-refractory colorectal cancer (CRC) were given MDAM at increasing dose levels from 80 to 300 mg/m2 per day intravenously for 5 days every 3 weeks. RESULTS: A total of 18 patients were entered into the study. Grade 2 or less nausea, vomiting, diarrhea, anorexia and fatigue were observed at doses > or =160 mg/m2 per day. Both patients enrolled at 300 mg/m2 per day experienced grade 3 stomatitis and one patient had grade 4 granulocytopenia. At 270 mg/m2 per day, grade 3 stomatitis (n=2), thrombocytopenia (n=1) and hyperbilirubinemia (n=1) were observed. All toxicities were relatively brief in duration and reversible. Leucovorin rescue was not required. Of 17 evaluable patients, no complete or partial responses were observed, and 3 patients demonstrated stable disease. Pharmacokinetic analyses were performed in 16 of the 18 patients receiving MDAM at doses of 80, 160, 240, 270 and 300 mg/m2. Normalized clearance of MDAM was approximately 1.5 times that reported for MTX (125 vs 80 ml/min per m2) in adults. CONCLUSION: MDAM is a novel antifolate with potential pharmacokinetic and safety advantages over MTX. Based on the results of this phase I study, stomatitis emerged as the dose-limiting toxicity and the recommended starting dose for phase II trials using this schedule and route of administration is 240 mg/m2 per day.