RESUMO
Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.
Assuntos
Aminopterina/análogos & derivados , Materiais Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Peptídeos/química , Temperatura , Aminopterina/química , Aminopterina/farmacologia , Materiais Biocompatíveis/química , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Géis/química , Humanos , Teste de MateriaisRESUMO
Since activated macrophages express a functional folate receptor ß (FRß), targeting this macrophage population with folate-linked drugs could increase selectivity to treat inflammatory diseases. Using a macrophage-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) in WKY rats, we investigated the effect of a novel folic acid-aminopterin (AMT) conjugate (EC2319) designed to intracellularly deliver AMT via the FR. We found that treatment with EC2319 significantly attenuated kidney injury and preserved renal function. Kidney protection with EC2319 was blocked by a folate competitor, indicating that its mechanism of action was specifically FRß-mediated. Notably, treatment with methotrexate (MTX), another folic acid antagonist related to AMT, did not protect from kidney damage. EC2319 reduced glomerular and interstitial macrophage infiltration and decreased M1 macrophage recruitment but not M2 macrophages. The expression of CCL2 and the pro-fibrotic cytokine TGF-ß were also reduced in nephritic glomeruli with EC2319 treatment. In EC2319-treated rats, there was a significant decrease in the deposition of collagens. In nephritic kidneys, FRß was expressed on periglomerular macrophages and macrophages present in the crescents, but its expression was not observed in normal kidneys. These data indicate that selectively targeting the activated macrophage population could represent a novel means for treating anti-GBM GN and other acute crescentic glomerulonephritis.
Assuntos
Receptor 2 de Folato/metabolismo , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/metabolismo , Aminopterina/química , Aminopterina/uso terapêutico , Animais , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Ácido Fólico/química , Ácido Fólico/uso terapêutico , Macrófagos/efeitos dos fármacos , Metotrexato/uso terapêutico , RatosRESUMO
One of the frontiers of nanomedicine is the rational design of theranostic nanovectors. These are nanosized materials combining diagnostic and therapeutic capabilities, i.e. capable of tracking cancer cells and tissues in complex environments, and of selectively acting against them. We herein report on the preparation and application of antifolate plasmonic nanovectors, made of functionalized gold nanoparticles conjugated with the folic acid competitors aminopterin and methotrexate. Due to the overexpression of folate binding proteins on many types of cancer cells, these nanosystems can be exploited for selective cancer cell targeting. The strong surface enhanced Raman scattering (SERS) signature of these nanovectors acts as a diagnostic tool, not only for tracing their presence in biological samples, but also, through a careful spectral analysis, to precisely quantify the amount of drug loaded on a single nanoparticle, and therefore delivered to the cells. Meanwhile, the therapeutic action is implemented based on the strong toxicity of antifolate drugs. Remarkably, supplying the drug in the nanostructured form, rather than as a free molecule, enhances its specific toxicity. The selectivity of the antifolate nanovectors can be optimized by the design of a hybrid folate/antifolate coloaded nanovector for the specific targeting of folate receptor α, which is overexpressed on numerous cancer cell types.
Assuntos
Antagonistas do Ácido Fólico/química , Nanoestruturas/química , Análise Espectral Raman , Nanomedicina Teranóstica , Aminopterina/química , Aminopterina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Fólico/química , Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Ouro/química , Humanos , Nanopartículas Metálicas/química , Metotrexato/química , Metotrexato/farmacologiaRESUMO
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.
Assuntos
Aminopterina/análogos & derivados , Antineoplásicos/uso terapêutico , Linfoma de Células T/tratamento farmacológico , Aminopterina/efeitos adversos , Aminopterina/química , Aminopterina/farmacocinética , Aminopterina/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , RecidivaRESUMO
Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N'-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.
Assuntos
Aminopterina/química , Anti-Inflamatórios/química , Ácido Fólico/análogos & derivados , Aminopterina/síntese química , Aminopterina/uso terapêutico , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/uso terapêutico , Desenho de Fármacos , Ácido Fólico/síntese química , Ácido Fólico/química , Ácido Fólico/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , EstereoisomerismoRESUMO
PURPOSE: On September 24, 2009, the U.S. Food and Drug Administration granted accelerated approval for Folotyn (pralatrexate injection, Allos Therapeutics, Inc.) as a single agent for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma (PTCL); it is the first drug approved for this indication. EXPERIMENTAL DESIGN: This review was based on study PDX-008, a phase II, single-arm, nonrandomized, open-label, international, multicenter trial, designed to evaluate the safety and efficacy of pralatrexate when administered concurrently with vitamin B(12) and folic acid supplementation in patients with relapsed or refractory PTCL. RESULTS: The overall response rate was 27% in 109 evaluable patients [95% confidence interval (CI), 19-36%]. Twelve percent of 109 evaluable patients (95% CI, 7-20%)] had a response duration of ≥14 weeks. Six of these 13 patients achieved a complete response, and one patient had complete response unconfirmed. The most common grade 3 and 4 toxicities were thrombocytopenia, mucositis, and neutropenia. CONCLUSION: This accelerated approval was based on a response rate that is reasonably likely to predict clinical benefit in this heavily pretreated patient population with this rare disease. The applicant has committed to conducting postmarketing clinical trials to assess clinical benefit. The recommended starting dose of pralatrexate in patients with relapsed or refractory PTCL is 30 mg/m(2) via intravenous push over 3 to 5 min weekly for 6 weeks followed by a one-week rest (one cycle). Intramuscular injection of 1 mg vitamin B(12) should be administered every 8 to 10 weeks along with 1.0 mg folic acid given orally once a day.
Assuntos
Aminopterina/análogos & derivados , Linfoma de Células T/tratamento farmacológico , Idoso , Aminopterina/efeitos adversos , Aminopterina/química , Aminopterina/uso terapêutico , Aprovação de Drogas , Feminino , Antagonistas do Ácido Fólico/efeitos adversos , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos , United States Food and Drug AdministrationRESUMO
PURPOSE: Renewed interest in antifols for the treatment of childhood cancers has resulted from identification of novel antifols with broad spectrums of anti-cancer activity and from re-evaluation of the original clinical antifol, aminopterin. In this pre-clinical study we evaluated the in vitro activity of both traditional antifols (methotrexate, aminopterin) and novel antifols (pemetrexed, talotrexin) in childhood acute leukemias and lymphomas. METHODS: We compared the in vitro cytotoxicity of methotrexate, aminopterin, pemetrexed, and talotrexin in a panel of six pediatric leukemia and lymphoma cell lines using the sulforhodamine B assay. In addition to defining a 50% growth inhibitory concentration (IC50) for a 120-h drug exposure, we contrasted the activity of the drugs in the context of clinically achievable (tolerable) drug exposures using the area under the plasma concentration-time curve (AUC). We defined each agent's clinical potency index (CPI) as the AUC achieved with standard pediatric dosing regimens divided by the in vitro IC50. RESULTS: Across all cell lines, talotrexin (median IC50 7 nM) and aminopterin (median IC50 17 nM) had lower IC50's than methotrexate (median IC50 78 nM) and pemetrexed (median IC50 155 nM). However, the CPI for methotrexate (median 0.9) was significantly greater than that for aminopterin (median 0.4). In contrast, pemetrexed had a significantly better CPI (median 13) than the traditional antifols. CONCLUSIONS: Aminopterin does not appear to offer any advantage over methotrexate for the treatment of childhood ALL. Further study of pemetrexed in childhood leukemias is warranted.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Aminopterina/química , Aminopterina/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Relação Dose-Resposta a Droga , Antagonistas do Ácido Fólico/química , Glutamatos/química , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/química , Guanina/farmacologia , Humanos , Concentração Inibidora 50 , Células Jurkat , Leucemia/patologia , Linfoma/patologia , Metotrexato/química , Metotrexato/farmacologia , Estrutura Molecular , Ornitina/análogos & derivados , Ornitina/química , Ornitina/farmacologia , Pemetrexede , Pterinas/química , Pterinas/farmacologiaRESUMO
4-Amino-4-deoxy-5,8,10-trideazapteroyl-d,l-4'-methyleneglutamic acid (CH-1504) is the prototype of a potentially therapeutically more selective class of antifolates for rheumatoid arthritis treatment. This class is characterized by retention of dihydrofolate reductase (DHFR; EC 1.5.1.3) as their locus of action and transport by the reduced folate carrier (RFC; SLC19A1), but their lack of metabolism by known pathways of antifolate (e.g., methotrexate (MTX)) metabolism. Five new CH-1504 analogs (CHL-001-CHL-005) were synthesized and diastereomers of CH-1504 itself were obtained by preparative chiral HPLC; all were characterized biochemically. The analogs are not metabolized by aldehyde oxidase (EC 1.2.3.1), carboxypeptidase G2 (EC 3.4.17.11), or (excepting CHL-003) folylpolyglutamate synthetase (EC 6.3.2.17) and thus, unlike MTX, are "metabolism-blocked". All analogs are potent DHFR inhibitors; several are nearly as potent as MTX or CH-1504. Each analog uses the RFC for transport, although with varying apparent affinities. In contrast, each weakly inhibits other enzymes of folate metabolism relevant to rheumatoid arthritis therapy (thymidylate synthase (EC 2.1.1.45), two formyltransferases of purine biosynthesis (EC 2.1.2.2 and EC 2.1.2.3), and 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20)). Biochemical characterization showed one 4'-diastereomer of racemic CH-1504 was significantly more active than the other. Based on literature data concerning the effect of d- and l-glutamic acid substitution on antifolate activity, it is likely that the diastereomer containing l-4'-methylene-glutamic acid is the more active. Because of concern about potential pharmacokinetic and biochemical effects of d-4'-methylene-glutamic acid-containing species, these data suggest that future analogs should contain only l-4'-methylene-glutamic acid. Overall, these data provide several interesting new leads for preclinical development.
Assuntos
Aminopterina/análogos & derivados , Antirreumáticos/uso terapêutico , Antagonistas do Ácido Fólico/uso terapêutico , Redes e Vias Metabólicas/fisiologia , Aminopterina/química , Aminopterina/farmacologia , Aminopterina/uso terapêutico , Animais , Antirreumáticos/química , Antirreumáticos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Linhagem Celular Tumoral , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Insetos , Redes e Vias Metabólicas/efeitos dos fármacos , CoelhosRESUMO
Ester prodrugs of the phosphinate pseudopeptide N-[(4-deoxy-4-amino-10-methyl)pteroyl]glutamate-gamma-[psiP(O)(OH)]-glutarate (1a) were synthesized. H-phosphinic acids derived from N-Cbz vinyl glycine esters were converted to the desired pseudopeptides by Michael addition to alpha-methyleneglutarate esters. Pivaloyloxymethyl (POM) ester moieties were incorporated in both the N-terminal and C-terminal fragments prior to formation of either C-P bond. N-Alkylation of the corresponding amides derived from p-(N-methyl)aminobenzoic acid with 2,4-diamino-6-(bromomethyl)pteridine gave the target compounds. POM esters of methotrexate and the corresponding gamma-glutamyl conjugate were also synthesized using the same strategy. All prodrugs were evaluated in Chinese hamster ovary cells. Although the pseudopeptide prodrugs were ineffective, prodrugs of methotrexate and the corresponding gamma-glutamyl conjugate were equipotent with the parent compounds. Stability of the prodrugs was investigated in both phosphate buffer and cell line medium to provide a rationale for the observed biological data.
Assuntos
Aminopterina/análogos & derivados , Antineoplásicos/síntese química , Peptídeo Sintases/antagonistas & inibidores , Ácidos Fosfínicos/síntese química , Pró-Fármacos/síntese química , Aminopterina/síntese química , Aminopterina/química , Aminopterina/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Hidrólise , Cinética , Metotrexato/administração & dosagem , Metotrexato/química , Metotrexato/farmacologia , Ácidos Pentanoicos/química , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-AtividadeRESUMO
Cyanobacterial and plant genomes encode proteins with some similarity to the folate and biopterin transporters of the trypanosomatid parasite Leishmania. The Synechocystis slr0642 gene product and its closest Arabidopsis homolog, the At2g32040 gene product, are representative examples. Both have 12 probable transmembrane domains, and the At2g32040 protein has a predicted chloroplast transit peptide. When expressed in Escherichia coli pabA pabB or folE, mutants, which are unable to produce or take up folates, the slr0642 protein and a modified At2g32040 protein (truncated and fused to the N terminus of slr0642) enabled growth on 5-formyltetrahydrofolate or folic acid but not on 5-formyltetrahydrofolate triglutamate, demonstrating that both proteins mediate folate monoglutamate transport. Both proteins also mediate transport of the antifolate analogs methotrexate and aminopterin, as evidenced by their ability to greatly increase the sensitivity of E. coli to these inhibitors. The full-length At2g32040 polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to the envelope of Arabidopsis chloroplasts in transient expression experiments. At2g32040 transcripts were present at similar levels in roots and aerial organs, indicating that the protein occurs in non-green plastids as well as chloroplasts. Insertional inactivation of At2g32040 significantly raised the total folate content of chloroplasts and lowered the proportion of 5-methyltetrahydrofolate but did not discernibly affect growth. These findings establish conservation of function among folate and biopterin transporter family proteins from three kingdoms of life.
Assuntos
Cianobactérias/metabolismo , Plastídeos/metabolismo , Trypanosoma/metabolismo , Aminopterina/química , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Transporte Biológico , Biopterinas/metabolismo , Membrana Celular/metabolismo , Clorofila/química , Cloroplastos/metabolismo , Clonagem Molecular , Sequência Conservada , Cianobactérias/química , Transportadores de Ácidos Dicarboxílicos/fisiologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Genoma de Planta , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Leucovorina/química , Metotrexato/farmacologia , Modelos Biológicos , Modelos Químicos , Mutação , Pisum sativum/metabolismo , Peptídeos/química , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/metabolismo , Synechocystis/metabolismoRESUMO
Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.
Assuntos
Aminopterina/farmacologia , Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Ornitina/análogos & derivados , Tetra-Hidrofolato Desidrogenase/metabolismo , Aminoácidos/química , Aminopterina/análogos & derivados , Aminopterina/química , Antineoplásicos/química , Ácidos Carboxílicos/química , Proteínas de Transporte/antagonistas & inibidores , Humanos , Ornitina/química , Ornitina/farmacologia , Pterinas/química , Pterinas/farmacologia , Proteína Carregadora de Folato Reduzido , Células Tumorais CultivadasRESUMO
The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate-albumin (MTX-SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT-SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT-SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT-SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT-SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight; p=0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT-SA was significantly more active. At the MTD/2 in W-256 carcinoma-bearing rats, AMPT-SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p=0.032). Tumor relapses were reduced and occurred later in the AMPT-SA group (two tumor recurrences for AMPT-SA versus seven for AMPT; p=0.05). In this comparative study, the AMPT-SA conjugate showed high antitumor activity in vivo and a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.
Assuntos
Aminopterina/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma 256 de Walker/tratamento farmacológico , Antagonistas do Ácido Fólico/uso terapêutico , Aminopterina/química , Aminopterina/toxicidade , Animais , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/toxicidade , Carcinoma 256 de Walker/diagnóstico por imagem , Quelantes , Sistemas de Liberação de Medicamentos , Feminino , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/toxicidade , Transplante de Neoplasias , Ácido Pentético , Cintilografia , Ratos , Ratos Sprague-Dawley , Albumina Sérica/química , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]-L-glutamic acid (1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl-L-glutamic acid dimethyl ester (6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester (2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine (7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.
Assuntos
Aminopterina/química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Aminopterina/farmacologia , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/síntese química , Humanos , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.
Assuntos
Antineoplásicos/farmacocinética , Antagonistas do Ácido Fólico/farmacocinética , Ornitina/análogos & derivados , Pterinas/farmacocinética , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Aminopterina/análogos & derivados , Aminopterina/química , Antineoplásicos/farmacologia , Sítios de Ligação , Divisão Celular , Resistência a Múltiplos Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Humanos , Ornitina/farmacocinética , Ornitina/farmacologia , Pterinas/farmacologia , Substâncias Redutoras , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
PURPOSE: These studies sought to evaluate the biochemical and cellular pharmacokinetic properties, cytotoxicity and antitumor efficacy of a new analogue of 10-deaza-aminopterin (PDX) against human tumors. METHODS: Studies were conducted with a group of human tumor cell lines in culture examining PDX and other folate analogues as permeants for mediated membrane transport, as inhibitors of dihdrofolate reductase and as substrates for folylpolyglutamate synthetase. These same analogues were examined for their cytotoxicity following a 3-h pulse exposure, in experiments providing a value for IC50. Other studies with these analogues were conducted in nude mice bearing subcutaneously implanted human tumors. Treatment of the mice was initiated 4 days after implantation of the tumor using a schedule of administration of one dose per day for 5 days. The tumors were measured 6 days after cessation of therapy and compared to controls for assessment of response. RESULTS: In the CCRF-CEM cell system, PDX was 2- to 3-fold less effective as an inhibitor of dihydrofolate reductase than aminopterin (AMT), methotrexate (MTX) or edatrexate (EDX) but much more effective as a permeant for one-carbon, reduced folate transport inward (PDX > AMT approximately equal to EDX > MTX) and substrate for folylpolyglutamate synthetase (PDX > AMT > EDX > MTX). As predicted by these results, PDX was 15- to 40-fold more cytotoxic than MTX and 3- to 4-fold more cytotoxic than the highly potent EDX following a 3-h pulse exposure in culture of CCRF-CEM cells and cells from a panel of three human breast and two human nonsmall-cell (NSC) lung cancers. The same relative differences were shown for the therapeutic efficacy of these three analogues at equitoxic doses in studies with the human MX-1 and LX-1 tumors and the human A549 NSC lung tumor xenografted in nude mice. On a schedule of qd x 5 given 3-4 days posttransplant, MTX was minimally active (modest tumor growth delay) against all three tumors. EDX was highly active (25-35% complete regressions and 5-10% cures) against the MX-1 and LX-1 tumors but very modestly active (no regressions) against the A549 tumor. In contrast, PDX was even more active (75-85% complete regressions and 25-30% cures) than EDX against the MX-1 and LX-1 tumors and highly active (30% complete regressions and 20% cures) against the A549 tumor. CONCLUSIONS: These studies showed significantly enhanced antitumor properties of PDX compared with MTX and EDX. Based upon these results, clinical trials of PDX in patients with metastatic breast and NSC lung cancer appear to be warranted.
Assuntos
Aminopterina/análogos & derivados , Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Aminopterina/química , Aminopterina/farmacologia , Animais , Antineoplásicos/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacocinética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metotrexato/farmacologia , Camundongos , Camundongos Nus , Relação Estrutura-Atividade , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Tumorais CultivadasAssuntos
Antineoplásicos , Indolquinonas , Taxoides , Aminopterina/análogos & derivados , Aminopterina/química , Antraquinonas/química , Antimetabólitos Antineoplásicos/química , Antineoplásicos/classificação , Antineoplásicos Alquilantes/química , Aziridinas/química , Hidrocarbonetos Aromáticos com Pontes/química , Briostatinas , Camptotecina/análogos & derivados , Camptotecina/química , Dacarbazina/análogos & derivados , Dacarbazina/química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Distamicinas/química , Doxorrubicina/química , Europa (Continente) , Humanos , Indóis/química , Irinotecano , Lactonas/química , Macrolídeos , Compostos de Mostarda Nitrogenada/química , Compostos de Nitrosoureia/química , Pirazóis/química , Quinazolinas/química , Temozolomida , Tiofenos/química , Alcaloides de Vinca/química , GencitabinaRESUMO
Twenty-six compounds derived from the 5-deaza- and 5,10-dideazaaminopterin series of aminopterin analogues were evaluated for antiarthritic activity in the mouse type II collagen model. New compounds in the 5-deaza series were prepared by alkylation of an appropriate N-substituted (4-aminobenzoyl)-L-glutamic acid dialkyl ester or N-(5-amino-2-thenoyl)-L-glutamate diester with a 2,4-diamino-5-alkyl-6-(bromomethyl)-5-deazapteridine. The resultant 5-deazaaminopterin diesters were saponified to provide the target 5-deaza analogues. 5,10-Dideazaaminopterins were synthesized by similar alkylation of the carbanions of appropriate 4-carboxyphenylacetic, (5-carboxy-2-thienyl)acetic, or (5-carboxy-2-pyridyl)acetic acid dimethyl esters. The diesters of the 2,4-diamino-4-deoxy-10-carboxy-5,10-dideazapteroic acid types so obtained were saponified and then readily decarboxylated by heating in Me2SO solution to provide the 2,4-diamino-5,10-dideazapteroic acid-type intermediates. Peptide coupling with diethyl L-glutamate followed by ester hydrolysis at room temperature afforded the new 5,10-dideazaaminopterin analogues. 5-Deazaaminopterins bearing an alkyl substituent at the 5-position were generally quite effective as antiinflammatory agents. Thus 5-propyl-5-deazaaminopterin, 5-methyl-10-propargyl-5-deazaaminopterin, 5-methyl-10-allyl-5-deazaaminopterin, 5-ethyl-5-deazamethotrexate, and 2,5-disubstituted thiophene analogue of 5-methyl-5-deazaaminopterin showed potencies greater than methotrexate by intraperitoneal or oral administration and were active over a considerably broader dose range. Useful activity in the 5,10-dideaza series was only observed for 5,10-dideazaaminopterin and its 10-methyl analogue. Alkyl substitution at C-5 or C-10 was generally detrimental to antiinflammatory activity in this series.
Assuntos
Aminopterina/análogos & derivados , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Metotrexato/análogos & derivados , Aminopterina/síntese química , Aminopterina/química , Aminopterina/farmacologia , Aminopterina/uso terapêutico , Animais , Antirreumáticos/síntese química , Antirreumáticos/química , Antirreumáticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colágeno , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Camundongos , Camundongos Endogâmicos DBA , Estrutura Molecular , Células Tumorais CultivadasRESUMO
10-Deaza modifications of classical antifolate analogues bearing the 1,4-disubstituted naphthalene ring in place of the 1,4-disubstituted benzene ring were prepared and tested for antitumor activity. Naphthalene analogues (9a-c, respectively) of 10-deazaaminopterin, 5-methyl-5, 10-dideazaaminopterin, and 5-ethyl-5,10-dideazaaminopterin were prepared by a route consisting of C-alkylations of the anion derived from 4-carboxyl-1-naphthaleneacetic acid dimethyl ester (2) by 6-(bromomethyl)-2,4-diaminopteridine (1a) and 6-(bromomethyl)-2,4-diamino-5-methyl- and -5-deazapteridines (1b and 1c, respectively) followed by ester hydrolysis and subsequent decarboxylation to give naphthalene analogues (7a-c, respectively) of 4-amino-4-deoxy-10-deazapteroic acid and 4-amino-4-deoxy-5- methyl- and -5-ethyl-5,10-dideazapteroic acids. Peptide coupling of 7a-c with L-glutamic acid dialkyl ester followed by mild ester hydrolysis gave target compounds 9a-c. The key advantage of this route is circumvention of a hydrogenation step requiring selectivity as in earlier approaches involving 9,10-olefinic precursors. Steric limitations thwarted plans to prepare the naphthalene analogue of 10-ethyl-10-deazaaminopterin; attempted alkylations of 2-(4-carboxy-1-naphthyl)butyric acid dimethyl ester with 1a failed as did attempted further alkylation (by EtBr) of the product derived from 1a and 2. Growth inhibition tests against three tumor cell lines (L1210, S180, and HL60) showed 9a to be 4-6-fold more inhibitory than methotrexate but not as inhibitory as 10-ethyl-10-deazaaminopterin; 9b and 9c were no more inhibitory than MTX. In tests against the EO771 mammary adenocarcinoma in mice, 9a was less active than MTX.
Assuntos
Aminopterina/análogos & derivados , Antineoplásicos/química , Adenocarcinoma/patologia , Alquilação , Aminopterina/química , Aminopterina/farmacologia , Animais , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Células Tumorais CultivadasRESUMO
Analogues of classical antifolates with the 4-aminobenzoyl group replaced by 4-amino-1-naphthoyl were synthesized for study after molecular modeling indicated ample spatial accommodation for the naphthalene ring and even larger groups in models based on reported X-ray crystallographic data describing the binding of methotrexate to human dihydrofolate reductase (DHFR). The side-chain precursors, N-(4-amino- and 4-(methylamino)-1-naphthoyl)-L-glutamic acid diethyl esters, were synthesized, and the 2,4-diamino-substituted heterocyclic groups were attached using several methods. Target compounds included naphthoyl analogues of aminopterin (AMT), methotrexate (MTX), 5-deazaAMT, 5-deazaMTX, 5-methyl-5-deazaAMT, 5-methyl-5-deazaMTX, and 5,8-dideazaAMT. A 5,6,7,8-tetrahydronaphthoyl analogue of 5-deazaAMT was also prepared. None of the naphthoyl analogues showed loss in binding to DHFR compared with the corresponding antifolate bearing the benzoyl group, thus confirming the anticipated bulk tolerance. Only the 5,6,7,8-tetrahydronaphthoyl analogue displayed reduced antifolate effects. Substrate activity toward folylpolyglutamate synthetase was, however, severely compromised. The naphthoyl compounds were transported into L1210 cells 3-6 times more readily than MTX, and despite apparently low levels of intracellular polyglutamylation, each compound was found to be significantly more potent than MTX in inhibiting tumor cell growth in vitro in three lines (L1210, HL60, and S180). The MTX, 5-methyl-5-deazaAMT, and 5-methyl-5-deazaMTX analogues were evaluated in vivo alongside MTX against E0771 mammary adenocarcinoma in mice. All three proved more effective than MTX in retarding the tumor growth. The naphthoyl analogue of 5-deazaAMT strongly inhibited DHFR from Pneumocystis carinii, Toxoplasma gondii, and rat liver giving IC50 (pM) values of 0.53, 2.1, and 1.6 respectively, but this compound did not inhibit in vitro growth of T. gondii, thus indicating lack of transport.
Assuntos
Benzoatos/química , Antagonistas do Ácido Fólico/síntese química , Naftalenos/química , Aminopterina/análogos & derivados , Aminopterina/química , Animais , Fenômenos Químicos , Físico-Química , Simulação por Computador , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Leucemia L1210/enzimologia , Fígado/enzimologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Metotrexato/análogos & derivados , Metotrexato/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Pneumocystis/enzimologia , Ratos , Relação Estrutura-Atividade , Toxoplasma/enzimologia , Células Tumorais CultivadasRESUMO
Recent development of new anticancer drugs in Japan was reviewed. Since there are a high incidence of cancer of the digestive organs, lung cancer and breast cancer in Japan, we have been looking for novel anticancer drugs effective against these solid cancers. Currently, some of fluoropyrimidine compounds, methotrexate analogues, tubulin inhibitors from plant origins have shown their clinical activities against human non-small cell lung cancer and/or breast cancer. As the end-point on these drug development, we have put an emphasis to find drugs to contribute for the survival prolongation and improvement of quality of life of the treated patients.