BACKGROUND & AIMS: :While pralatrexate (PDX) has been successfully developed for the treatment of T-cell lymphoma, the mechanistic basis for its T-cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX-resistant cell lines H9-12 and H9-200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9-12 and H9-200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration-dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9-12 and H9-200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.

背景与目标： : 尽管已成功开发了pralatrexate (PDX) 用于治疗T细胞淋巴瘤，但其T细胞选择性和获得性耐药性的机制基础仍然难以捉摸。为了潜在地识别可能绕过或延迟获得的PDX抗性的协同组合，我们在宽浓度范围内产生了抗性细胞系。开发了PDX抗性细胞系H9-12和H9-200，分别表现出35和超过1000  nM的IC50。这些品系是从亲本H9细胞体外建立的。对已知是抗叶酸药理学重要决定因素的蛋白质的表达分析表明，由于基因扩增，二氢叶酸还原酶 (DHFR) 的表达增加，叶酸载体1的下调减少，这是H9-12和H9-200细胞耐药的推定机制。交叉耐药性仅在甲氨蝶呤中可见，而在罗米丁，阿扎胞苷 (AZA)，地西他滨，吉西他滨，阿霉素或硼替佐米中则未见。通过以浓度依赖的方式用次甲基化剂预处理，可以逆转对PDX的抗性。亲本和耐药细胞系基因表达谱的比较证实了基因表达模式的明显不同，并确定了双特异性磷酸酶四 (DUSP4) 是PDX活性的分子靶标之一。在H9中观察到暴露于PDX后STAT5磷酸化降低，但在H9-12和H9-200细胞中未观察到。这些数据表明，与低甲基化剂的组合可能是有效的，并且DUSP4和STAT5可能代表PDX活性的假定生物标志物。

BACKGROUND & AIMS: BACKGROUND:Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents. METHODS:Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT-PCR. RESULTS:Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance. CONCLUSION:Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome.

背景与目标：

BACKGROUND & AIMS: PURPOSE:Pralatrexate is a rationally designed antifolate with greater preclinical antitumor activity than methotrexate. Pralatrexate was synergistic with paclitaxel and with docetaxel in mouse xenograft experiments. This phase 1 study was designed to determine the maximum tolerated dose and toxicity of pralatrexate plus paclitaxel or docetaxel in patients with advanced cancer. EXPERIMENTAL DESIGN:Pralatrexate was administered i.v. every 2 weeks (days 1 and 15) in a 4-week cycle. Depending on the taxane used and dose being tested, the taxane was administered on days 1 and 15; days 2 and 16; or days 1, 8, and 15. In the latter part of the study, patients in the docetaxel arm were treated with vitamin B(12) and folic acid supplementation to mitigate toxicity and allow pralatrexate dose escalation. RESULTS:For the combination of pralatrexate plus paclitaxel without vitamin supplementation, dose-limiting stomatitis and peripheral neuropathy were encountered at the lowest dose levels tested. For pralatrexate plus docetaxel plus vitamin supplementation, pralatrexate 120 mg/m(2) plus docetaxel 35 mg/m(2) administered on the same day every other week was defined as the maximum tolerated dose and schedule, with dose-limiting toxicities at higher dose combinations including stomatitis and asthenia. Significant antitumor activity was observed for this combination in patients with non-small-cell lung cancer. CONCLUSIONS:Pralatrexate (120 mg/m(2)) plus docetaxel (35 mg/m(2)) plus vitamin supplementation is well tolerated with signs of efficacy against non-small-cell lung cancer that merit phase 2 testing.

背景与目标：

BACKGROUND & AIMS: 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.

背景与目标：

BACKGROUND & AIMS: -2

背景与目标： -2

BACKGROUND & AIMS: PURPOSE:This study evaluated mechanistic differences of pralatrexate, methotrexate, and pemetrexed. METHODS:Inhibition of dihydrofolate reductase (DHFR) was quantified using recombinant human DHFR. Cellular uptake and folylpolyglutamate synthetase (FPGS) activity were determined using radiolabeled pralatrexate, methotrexate, and pemetrexed in NCI-H460 non-small cell lung cancer (NSCLC) cells. The tumor growth inhibition (TGI) was assessed using MV522 and NCI-H460 human NSCLC xenografts. RESULTS:Apparent K ( i ) values for DHFR inhibition were 45, 26, and >200 nM for pralatrexate, methotrexate, and pemetrexed, respectively. A significantly greater percentage of radiolabeled pralatrexate entered the cells and was polyglutamylatated relative to methotrexate or pemetrexed. In vivo, pralatrexate showed superior anti-tumor activity in both NSCLC models, with more effective dose-dependent TGI in the more rapidly growing NCI-H460 xenografts. CONCLUSIONS:Pralatrexate demonstrated a distinct mechanistic and anti-tumor activity profile relative to methotrexate and pemetrexed. Pralatrexate exhibited enhanced cellular uptake and increased polyglutamylation, which correlated with increased TGI in NSCLC xenograft models.

背景与目标：

BACKGROUND & AIMS: :Pralatrexate inhibits folic acid metabolism, and preclinical studies have shown that it is cytotoxic to multiple myeloma cells. This phase 1 study investigated the safety and efficacy of pralatrexate in combination with bortezomib in adults with relapsed or refractory multiple myeloma. A standard 3 + 3 design was used. Patients received intravenous pralatrexate at doses ranging from 10 to 30 mg/m(2) and intravenous bortezomib at a dose of 1·3 mg/m(2) on days 1, 8 and 15 of each 4-week cycle. Eleven patients were enrolled and completed a median of two cycles. The maximum tolerated dose was 20 mg/m(2) . Two patients experienced dose-limiting toxicity of mucositis. The most frequent non-haematological toxicities were fatigue (55%) and mucositis (45%). There were three serious adverse events in three patients: rash, sepsis and hypotension. One patient (9%) had a very good partial response, 1 (9%) had a partial response, 1 (9%) had minimal response and two (18%) had progressive disease. The median duration of response was 4 months, the median time to next treatment was 3·4 months and the median time to progression was 4 months. Pralatrexate, in combination with bortezomib, was generally safe and demonstrated modest activity in relapsed or refractory multiple myeloma. Clinicaltrials.gov identifier: NCT01114282.

背景与目标： : Pralatrexate抑制叶酸代谢，临床前研究表明它对多发性骨髓瘤细胞具有细胞毒性。这项1期研究调查了普拉瑞沙与硼替佐米联合治疗成人复发性或难治性多发性骨髓瘤的安全性和有效性。使用标准3 3设计。患者在每个4周周期的第1、8和15天接受10至30 mg/m(2) 的静脉注射普拉托雷酯和1·3 mg/m(2) 的静脉注射硼替佐米。11例患者入选并完成了两个周期的中位。最大耐受剂量为20 mg/m(2)。两名患者经历了粘膜炎的剂量限制性毒性。最常见的非血液学毒性是乏力 (55%) 和粘膜炎 (45%)。3例患者发生了3例严重不良事件: 皮疹、败血症和低血压。一名患者 (9%) 具有非常好的部分反应，1名患者 (9%) 具有部分反应，1名患者 (9%) 具有最小反应，两名患者 (18%) 具有进行性疾病。中位缓解持续时间为4个月，至下一次治疗的中位时间为3·4个月，中位进展时间为4个月。Pralatrexate联合硼替佐米通常是安全的，并且在复发或难治性多发性骨髓瘤中表现出适度的活性。临床医生.Gov标识符: nct01114282。

BACKGROUND & AIMS: :It has been nearly 8 years since pralatrexate became the first drug approved by the U.S. Food and Drug Administration for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL). Like most drugs approved for a particular clinical indication, as much or more is learned once it enters mainstream use as in the years leading up to regulatory approval. Over the past several years, many diverse lines of research have shed new insight into both the agent, and the diseases it treats. In this review, we will bring the reader up to date on the many new aspects related to pralatrexate's pharmacology, activity across the panoply of T-cell lymphoproliferative malignancies, as well as some new and emerging guidelines that are likely to improve its safety profile. Finally, the review will close with the many new lines of evidence building a rationale for the combination of these novels: novel combination, and the vision for new platforms in PTCL care.

背景与目标： : 自普拉曲酯成为美国食品药品监督管理局批准的第一种用于治疗复发性或难治性外周T细胞淋巴瘤 (PTCL) 的药物以来，已有近8年的历史。像大多数被批准用于特定临床适应症的药物一样，一旦进入主流应用，就会学到更多或更多，就像在获得监管部门批准之前的几年一样。在过去的几年中，许多不同的研究领域对药物及其治疗的疾病都有了新的认识。在这篇综述中，我们将向读者介绍与普拉雷斯特的药理学，整个T细胞淋巴增生性恶性肿瘤的活动有关的许多新方面，以及一些可能改善其安全性的新指南。最后，审查将以许多新的证据为结束，这些小说的结合建立了基础: 小说的结合以及PTCL护理新平台的愿景。

BACKGROUND & AIMS: 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.

背景与目标：

BACKGROUND & AIMS: :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.

背景与目标： : Pralatrexate是一种叶酸类似物代谢抑制剂，由Spectrum Pharmaceuticals，Inc.的全资子公司Allos Therapeutics，Inc.生产。在临床前和临床研究中，普拉酯在淋巴瘤中均具有活性。Pralatrexate已被美国FDA批准用于治疗复发/难治性外周T细胞淋巴瘤2009年。批准是基于PROPEL试验的数据，该试验证明在经过大量预处理的患者人群中总体缓解率为29%。pralatrexate的剂量和时间表为每周30 mg/m(2)，持续6周，以7周为周期。需要补充叶酸和维生素B12以最大程度地减少毒性。最常见的毒性是粘膜炎，血小板减少症，恶心和乏力。

BACKGROUND & AIMS: BACKGROUND:Several previous clinical trials have shown that malignant pleural mesothelioma is responsive to antifolates. The dihydrofolate reductase inhibitor, pralatrexate, has a favorable toxicity profile, primarily limited to stomatitis, and has demonstrated activity in patients with non-small cell lung cancer. In mesothelioma cell lines and xenografts, pralatrexate demonstrated significant antitumor activity. METHODS:We conducted this phase II study to determine the response rate of malignant pleural mesothelioma to pralatrexate at a dose of 135 mg/m2 i.v. every 2 weeks. After a protocol amendment, patients were supplemented with vitamin B12 and folic acid at the time of starting therapy. RESULTS:A total of 16 assessable patients were enrolled. No complete or partial responses were observed. Two patients with epithelioid histology had minor responses. Three other patients remained on study with stable disease for 9, 9, and 48 months. The median time to progression was 3 months. The overall median survival time was 7 months (95% confidence interval: 3.2-16.2 months) and the one-year survival was 31% (95% confidence interval: 15%-65%). Three patients (19%) had grade 2 stomatitis, eight (50%) had grade 3, and one (6%) had grade 4. CONCLUSIONS:With this particular dose and schedule, pralatrexate as a single agent had no activity in malignant pleural mesothelioma.

背景与目标：

BACKGROUND & AIMS: 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.

背景与目标：

BACKGROUND & AIMS: :Here we report on the treatment of a 38-year-old woman with primary cutaneous γδT-cell lymphoma, which is a rare subset of cutaneous T-cell lymphoma. She presented with multiple subtle subcutaneous nodules, which were not clearly observed on computed tomography scans or after biopsy. However, F-fluorodeoxyglucose positron emission tomography (F-FDG-PET) accurately detected small cutaneous lesions. She achieved a second complete remission, as demonstrated by F-FDG-PET performed after pralatrexate infusion.

背景与目标： : 在这里，我们报道了一名38岁女性原发性皮肤 γ δ T细胞淋巴瘤的治疗，这是皮肤T细胞淋巴瘤的罕见子集。她出现了多个细微的皮下结节，在计算机断层扫描或活检后均未清楚观察到。但是，F-氟脱氧葡萄糖正电子发射断层扫描 (f-fdg-pet) 准确地检测到了小的皮肤病变。如pralatrexate输注后进行的f-fdg-pet所示，她获得了第二次完全缓解。

BACKGROUND & AIMS: IMPORTANCE OF THE FIELD:Pralatrexate (PDX; 10-propargyl 10-deazaaminopterin), is an exciting new chemotherapeutic agent that is approved for the treatment of relapsed and refractory peripheral T-cell lymphomas (PTCL). This is the only approved therapy for patients with PTCL. AREAS COVERED IN THIS REVIEW:This review describes the clinical development of PDX from its synthesis to its FDA approval. It details the biochemical basis of the differences between PDX and other antifolates that form the basis of the superiority of its activity. This is followed by a description of the preclinical data that led to early-phase clinical trials in lung cancer and lymphoma and, finally, the definitive trial that led to its approval in PTCL. The review also describes how PDX is being combined with other agents in both the preclinical and clinical arenas. WHAT THE READER WILL GAIN:These trials have been instrumental in defining a safe dose as well as the safety profile of the agent. FDA approval for the use of PDX in PTCL has been granted based on the results of the pivotal Phase II trial of this agent in relapsed and refractory PTCL patients. TAKE HOME MESSAGE:PDX is a unique antifolate that has been rationally designed to have a high affinity for the reduced folate receptor (RFC-1) and the enzyme folylpolyglutamy synthetase. It is active in T-cell lymphomas and non-small-cell lung cancer. It is now being studied in combination with other chemotherapeutic and targeted agents for the treatment of hematological malignancies.

背景与目标：

BACKGROUND & AIMS: INTRODUCTION:Pralatrexate, a folate analogue targeting dihydrofolate reductase, has antitumor activity in non-small-cell lung cancer (NSCLC). This randomized phase 2b trial was designed to further evaluate pralatrexate activity in NSCLC by estimating overall survival (OS) relative to erlotinib in patients with relapsed/refractory disease. METHODS:In 43 centers across 6 countries, patients were randomized 1:1 to receive intravenous pralatrexate 190 mg/m on days 1 and 15 of a 28-day cycle, or oral erlotinib 150 mg/day. The primary objective was to estimate OS in all patients and prespecified subgroups using relative comparisons of hazard ratios (HRs). Secondary endpoints included progression-free survival, response rate, and safety. Key eligibility criteria included: (1) ≥1 prior platinum-based therapy, (2) Eastern Cooperative Oncology Group performance status of 0 to 1, and 3) a smoking history of 100 cigarettes or more. RESULTS:A total of 201 patients were randomized. A trend toward improvement in OS favoring pralatrexate was observed with an HR of 0.84 (95% confidence interval: 0.61-1.14) in the intent-to-treat population. This favorable survival result was seen in most prespecified subgroups for pralatrexate. The largest reduction in the risk of death was observed in patients with nonsquamous cell carcinoma (n = 107; HR = 0.65; 95% confidence interval: 0.42-1.0). The most common grade 3 to 4 adverse event in the pralatrexate arm was mucositis (23%). Discontinuation of pralatrexate for any grade of mucositis was 21%. CONCLUSIONS:Pralatrexate demonstrated a trend toward improved survival relative to erlotinib in patients with advanced NSCLC. Future studies should include a mucositis management plan to improve tolerability and maximize treatment benefit.

背景与目标：