RESUMEN
Cladribine is a purine nucleoside analog initially developed in the 1970s as a treatment for various blood cancers. Due to the molecule's ability to preferentially reduce T and B lymphocytes, it has been developed into an oral formulation for the treatment of multiple sclerosis (MS). The unique proposed mechanism of action of cladribine allows for the therapy to be delivered orally over two treatment-week cycles per year, one cycle at the beginning of the first month and one cycle at the beginning of the second month of years 1 and 2, with the potential for no further cladribine treatment required in years 3 and 4. This review summarizes the clinical development program for cladribine tablets in patients with MS, including the efficacy endpoints and results from the 2-year phase III CLARITY study in patients with relapsing-remitting MS (RRMS), the 2-year CLARITY EXTENSION study, and the phase III ORACLE-MS study in patients with a first clinical demyelinating event at risk for developing MS. Efficacy results from the phase II ONWARD study, in which cladribine tablets were administered as an add-on to interferon-ß therapy in patients with RRMS, are also summarized. A review of all safety data, including lymphopenia, infections, and malignancies, is provided based on data from all trials in patients with MS, including the initial parenteral formulation studies. Based on these data, cladribine tablets administered at 3.5 mg/kg over 2 years have been approved across the globe for various forms of relapsing MS. The development of cladribine tablets for the treatment of multiple sclerosis: a comprehensive review (MP4 279143 kb).
Asunto(s)
Cladribina/química , Cladribina/uso terapéutico , Inmunosupresores/química , Inmunosupresores/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Comprimidos/química , Comprimidos/uso terapéutico , Química Farmacéutica/métodos , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , HumanosRESUMEN
Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy-cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled-up reaching a productivity of 138 mg L-1 hr-1 . Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions.
Asunto(s)
Alginatos/química , Antineoplásicos/metabolismo , Cladribina/metabolismo , Micrococcaceae/metabolismo , Antineoplásicos/química , Biocatálisis , Biotransformación , Cladribina/químicaRESUMEN
The combination of cytarabine and an anthracycline has been the standard of care for the induction of remission in acute myeloid leukemia (AML). The response to treatment and survival of adult patients with AML are still variable and depend on multiple factors. Therefore, there have been many efforts to improve the response to treatment and survival rates by either increasing the cytarabine dose or adding a third agent to the standard induction chemotherapy regimen. Unfortunately, attempts to improve response and survival have been mostly unsuccessful. Recent clinical trials and retrospective studies explored the addition of cladribine to standard induction chemotherapy for AML. Some of these studies showed higher rates of complete remission, and one showed improved survival. In this review, we will discuss the antileukemic properties of cladribine and summarize the recent clinical data regarding its incorporation into the induction therapy for adult AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cladribina , Leucemia Mieloide Aguda/tratamiento farmacológico , Antraciclinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Cladribina/administración & dosificación , Cladribina/efectos adversos , Cladribina/química , Cladribina/farmacocinética , Ensayos Clínicos como Asunto , Citarabina/administración & dosificación , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Cardiopatías/inducido químicamente , Enfermedades Hematológicas/inducido químicamente , Humanos , Quimioterapia de Inducción , Liposomas , Metaanálisis como Asunto , Mucositis/inducido químicamente , Estudios Multicéntricos como Asunto , Neoplasias Primarias Secundarias/inducido químicamente , Inducción de Remisión , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Cladribine (2-CdA) is used as an anti-cancer drug but is currently studied as a potential treatment for use in relapsing-remitting multiple sclerosis (MS). In this study, we computer designed, synthesized, and characterized two novel derivatives of 2-CdA, K1-5d and K2-4c, and investigated their underlying mechanism of beneficial effect using the CCRF-CEM and RAJI cell lines. For this purpose, we first determined their effect on MS and DNA damage and repair-related gene expression profiles using custom arrays along with 2-CdA treatment at non-toxic doses. Then, we determined whether cells underwent apoptosis after treatment with 2-CdA, K1-5d, and K2-4c in CCRF-CEM and RAJI cells, using the DNA fragmentation assay. It was found that both derivatives modulated the expression of the pathway-related genes that are important in inflammatory signaling, apoptosis, ATM/ATR, double-strand break repair, and the cell cycle. Furthermore, 2-CdA, K1-5d, and K2-4c significantly activated apoptosis in both cell lines. In summary, our data demonstrate that although both derivatives act as anti-inflammatory and apoptotic agents, inducing the accumulation of DNA strand breaks and activating the ultimate tumor suppressor p53 in T and B lymphocytes, the K1-5d derivative has shown more promising activities for further studies.
Asunto(s)
Cladribina/farmacología , Daño del ADN/efectos de los fármacos , Inmunosupresores/farmacología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Cladribina/síntesis química , Cladribina/química , Simulación por Computador , Roturas del ADN/efectos de los fármacos , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/química , Simulación del Acoplamiento Molecular , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Chlorinase SalL halogenate S-adenosyl-l-methionine (SAM) reacts with chloride to generate 5'-chloro-5'-deoxyadenosine and l-methionine through a nucleophilic substitution mechanism. Although it is known that chlorinase enhances the rate of reaction by a factor of 1.2 × 1017 fold, it is not entirely clear how this is accomplished. The search for the origin of the catalysis of chlorinase and other enzymes has led to a desolvation hypothesis. In the present work, we have used well defined computational simulations in order to evaluate the origin of the catalytic efficiency of chlorinase. The results demonstrate that the catalytic effect of chlorinase is associated with the fact that Cl- is "solvated" by the protein more than by the reference solution reaction, which is not in accordance with proposed catalysis by desolvation. It is found that chlorinase SalL active sites provide electrostatic stabilization of the transition state which is the origin of its catalytic effect.
Asunto(s)
Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Biocatálisis , Dominio Catalítico , Cladribina/química , Cladribina/metabolismo , Enlace de Hidrógeno , Metionina/química , Metionina/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Mutagénesis Sitio-Dirigida , S-Adenosilmetionina/química , Electricidad Estática , Termodinámica , Agua/químicaRESUMEN
Antimetabolite drugs, including the adenosine deaminase inhibitor cladribine, have been shown to induce apoptosis in a variety of cancer cells, and have been widely used in clinical trials of various cancers in conjunction with tyrosine kinase inhibitors (TKIs). Combination treatment with cladribine and gefitinib or dasatinib is expected to have a synergistic inhibitory effect on breast cancer cell growth. Our results demonstrated that the combination treatment had synergistic activity against human breast cancer (MCF-7) cells, enhanced G2/M cell arrest and reactive oxygen species (ROS) generation, and increased the loss of mitochondrial membrane potential and cell apoptosis. In addition, the combination treatment decreased Bcl-2 expression. Our results demonstrated that cladribine in combination with gefitinib or dasatinib exerted synergistic anticancer effects on MCF-7 cells by inducing cell cycle arrest, ROS production and apoptosis through the mitochondria-mediated intrinsic pathway.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Cladribina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cladribina/síntesis química , Cladribina/química , Dasatinib/química , Dasatinib/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Gefitinib , Humanos , Células MCF-7 , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/química , Quinazolinas/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
Molecular determinants of FlA1 fluorinase specificity were probed using 5'-chloro-5'-deoxyadenosine (5'-ClDA) analogs as substrates and FlA1 active site mutants. Modifications at F213 or A279 residues are beneficial towards these modified substrates, including 5'-chloro-5'-deoxy-2-ethynyladenosine, ClDEA (>10-fold activity improvement), and conferred novel activity towards substrates not readily accepted by wild-type FlA1.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sondas Moleculares/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Cladribina/química , Cladribina/metabolismo , Modelos Moleculares , Sondas Moleculares/química , Mutación , Conformación de Ácido Nucleico , Oxidorreductasas/química , Streptomyces/enzimología , Especificidad por SustratoRESUMEN
The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2ß2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.
Asunto(s)
Biopolímeros/química , Cladribina/química , Nucleótidos/química , Ribonucleótido Reductasas/antagonistas & inhibidores , Vidarabina/análogos & derivados , Cromatografía en Gel , Polarización de Fluorescencia , Vidarabina/químicaRESUMEN
New insight into the in vitro and in vivo metabolism of Cladribine (2-chloro-2'-deoxyadenosine, [2-CdA]) are presented. Following incubation of [(14)C]-2-CdA in mouse, rat, rabbit, dog, monkey and human hepatocyte cultures, variable turnover was observed with oxidations and direct glucuronidation pathways. The oxidative cleavage to 2-chloroadenine (2-CA, M1) was only observed in rabbit and rat. Following incubation of [(14)C]-2-CdA in whole blood from mouse, monkey and human, a significant turnover was observed. The main metabolites in monkey and human were 2-chlorodeoxyinosine (M11, 16% of total radioactivity) and 2-chlorodeoxyinosine (M12, 43%). In mouse, 2-CA was the major metabolite (2-CA; M1, 73%). After single intravenous and oral administration of [(14)C]-2-CdA to mice, 2-chlorodeoxyinosine (M11) was confirmed in plasma, while 2-chlorohypoxanthine (M12) and 2-CA (M1) were found in urine. Overall, the use of [(14)C]-2-CdA both in vitro (incubations in mouse, monkey and human whole blood) and in vivo (mouse) has confirmed the existence of an additional metabolism pathway leading to the formation of 2-chlorodeoxyinosine (M11) and 2-chlorohypoxanthine (M12). Formation of these two metabolites demonstrates that Cladribine as free form is not fully resistant to adenosine deaminase as suggested earlier, an enzyme involved in its mode of action.
Asunto(s)
Cladribina/metabolismo , Administración Intravenosa , Administración Oral , Animales , Cromatografía Líquida de Alta Presión , Cladribina/sangre , Cladribina/química , Cladribina/orina , Perros , Heces , Haplorrinos , Hepatocitos/metabolismo , Humanos , Espectrometría de Masas , Redes y Vías Metabólicas , Metaboloma , Ratones , Conejos , RatasRESUMEN
Multiple sclerosis (MS) often results in chronic inflammatory and autoimmune disorders, and recent developments in understanding the disease pathogenesis has lead to newer therapeutic options for the treatment of the disease. The development of small molecule drugs with improved efficacy, better tolerability, and oral administration has received a new impetus with the discovery of newer classes of drugs. In this review, we have summarized the hitherto known synthetic strategies of fingolimod, laquinimod, cladribine, and teriflunomide reported in the literature which are the key small molecules and the first oral drug candidates for MS in various stages of clinical development or have been launched in the market.
Asunto(s)
Cladribina/uso terapéutico , Crotonatos/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Glicoles de Propileno/uso terapéutico , Quinolonas/uso terapéutico , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Esfingosina/análogos & derivados , Toluidinas/uso terapéutico , Cladribina/síntesis química , Cladribina/química , Crotonatos/síntesis química , Crotonatos/química , Clorhidrato de Fingolimod , Humanos , Hidroxibutiratos , Estructura Molecular , Nitrilos , Glicoles de Propileno/síntesis química , Glicoles de Propileno/química , Quinolonas/síntesis química , Quinolonas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Esfingosina/síntesis química , Esfingosina/química , Esfingosina/uso terapéutico , Toluidinas/síntesis química , Toluidinas/químicaRESUMEN
INTRODUCTION: Multiple sclerosis (MS) is a chronic immune-mediated disorder of the central nervous system leading to progressive neurodegeneration and disability. Until 2010, all approved disease-modifying drugs for MS required parenteral administration, which is associated with suboptimal adherence. It was anticipated that new approaches to treatment, including oral agents such as cladribine tablets, may improve adherence. In 2011, the development of cladribine tablets was stopped following negative feedback from the EMA and FDA. AREAS COVERED: This article provides an overview of the chemistry, mechanism of action and pharmacological properties of cladribine tablets therapy, and highlights the rationale for its development as an oral treatment for MS. Key efficacy and safety data from the pivotal Phase III CLARITY study are presented, providing context for the opinion received from the regulatory agencies. EXPERT OPINION: Despite the promising efficacy data observed in the cladribine tablets clinical trial program, regulatory agencies identified a potential risk of increased malignancies, and raised concerns about the implications of sustained lymphocyte depletion. Following the feedback received from the regulatory agencies, Merck Serono made the decision to withdraw the agent from the regulatory approval process. The experience gained will benefit other research efforts to address the outstanding unmet treatment needs of patients with relapsing MS.
Asunto(s)
Cladribina/uso terapéutico , Inmunosupresores/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Administración Oral , Cladribina/química , Cladribina/farmacocinética , Ensayos Clínicos Fase III como Asunto , Femenino , Humanos , Inmunosupresores/química , Inmunosupresores/farmacocinética , Linfocitos/efectos de los fármacos , Masculino , Retirada de Medicamento por Seguridad , ComprimidosRESUMEN
Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.
Asunto(s)
Antineoplásicos/metabolismo , Desoxicitidina Quinasa/metabolismo , Nucleósidos de Purina/metabolismo , Nucleósidos de Pirimidina/metabolismo , Serina/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Afidicolina/farmacología , Biotransformación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cladribina/química , Cladribina/metabolismo , Cladribina/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Desoxicitidina Quinasa/antagonistas & inhibidores , Desoxicitidina Quinasa/genética , Activación Enzimática , Células HCT116 , Células HT29 , Humanos , Cinética , Mutación , Fosforilación , Nucleósidos de Purina/química , Nucleósidos de Purina/farmacología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Serina/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Vidarabina/análogos & derivados , Vidarabina/química , Vidarabina/metabolismo , Vidarabina/farmacología , GemcitabinaRESUMEN
A new efficient route for the synthesis of 2-chloro-2';-deoxyadenosine (Cladribine), CdA) has been developed. The key step of this method was selective deprotection of the acetyl group at the 2' position; the 3', 5' acetyl groups were not affected. This can be accomplished efficiently with hydroxylamine hydrochloride and sodium acetate in pyridine. The 2' hydroxyl group was removed by the Barton-McCombie reaction. Using this strategy, CdA was prepared in five steps and 31.0% yields.
Asunto(s)
Antineoplásicos/síntesis química , Cladribina/síntesis química , Antineoplásicos/química , Cladribina/químicaRESUMEN
Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.
Asunto(s)
Antineoplásicos/uso terapéutico , Cladribina/uso terapéutico , Linfoma de Células B/tratamiento farmacológico , Proteoma/análisis , Proteoma/efectos de los fármacos , Purinas/farmacología , Vidarabina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Cladribina/química , Cladribina/farmacología , Electroforesis en Gel Bidimensional , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , Linfoma de Células B/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Purinas/química , Vidarabina/química , Vidarabina/farmacología , Vidarabina/uso terapéuticoRESUMEN
Cladribine, a synthetic deoxyadenosine analog, is an oral immunomodulatory agent that produces targeted, sustained reduction of T and B lymphocytes. This mechanism of action provides the rationale for use in relapsing-remitting multiple sclerosis (MS) in a short-course annual dosing regimen. Based on the results of a pivotal Phase III study, therapy with cladribine tablets has the potential to become a licensed oral disease-modifying medication for relapsing forms of MS. This article will review the key points regarding MS and its pathogenesis, and discuss current unmet treatment needs. In particular the review provides an overview of emerging potential new oral MS therapies with a focus on the mechanism of action, chemistry, pharmacokinetics, pharmacodynamics, clinical efficacy and safety of cladribine tablets. Assessments and conclusions will include a speculative 5-year outlook.
Asunto(s)
Cladribina/uso terapéutico , Inmunosupresores/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Administración Oral , Cladribina/química , Ensayos Clínicos Fase III como Asunto , Humanos , Esclerosis Múltiple Recurrente-Remitente/patología , Vigilancia de Productos ComercializadosRESUMEN
Cladribine was synthesized as a purine analogue drug that inhibited adenosine deaminase. It received FDA approval in the 1980s for treatment of hairy cell leukemia. Given its toxicity towards lymphocytes and its corresponding immunosuppressive effects, it has been studied and found efficacious in a variety of hematologic malignancies and autoimmune conditions, most recently multiple sclerosis. This review highlights pharmacological, toxicological and clinical data for the use of cladribine. It also discusses existing and new mechanisms that may contribute to its unique clinical activity. Emerging data show that in addition to its known purine nucleoside analogue activity, cladribine possesses epigenetic properties, inhibiting S-adenosylhomocysteine hydrolase and DNA methylation. This may contribute to its efficacy and highlights the importance of studying combination therapy with other epigenetic or targeted agents. Clinical trials are underway in a variety of malignant and nonmalignant conditions.
Asunto(s)
Inhibidores de la Adenosina Desaminasa , Antineoplásicos/farmacología , Cladribina/farmacología , Purinas/química , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Cladribina/efectos adversos , Cladribina/química , Cladribina/farmacocinética , Cladribina/uso terapéutico , Ensayos Clínicos como Asunto , Metilación de ADN/efectos de los fármacos , Humanos , Estructura Molecular , Neoplasias/tratamiento farmacológicoRESUMEN
Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.
Asunto(s)
Cladribina/metabolismo , Lactonas/metabolismo , Sintasas Poliquetidas/metabolismo , Pirroles/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Cladribina/química , Clonación Molecular , Orden Génico , Genoma Bacteriano/genética , Cinética , Lactonas/química , Malonil Coenzima A/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Estructura Molecular , Familia de Multigenes , Mutación , Filogenia , Sintasas Poliquetidas/genética , Pirroles/química , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylated at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.
Asunto(s)
Desoxicitidina Quinasa/metabolismo , Nucleósidos de Purina/metabolismo , Sitios de Unión , Cladribina/química , Cristalografía por Rayos X , Desoxicitidina Quinasa/química , Desoxicitidina Quinasa/genética , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación ProteicaRESUMEN
The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) was examined with various ATP analogues of clinical relevance. The triphosphate derivatives of cladribine (2-Cl-dATP), clofarabine (Cl-F-ara-ATP), fludarabine (F-ara-ATP), and related derivatives were incubated with yPAP and 32P-radiolabeled RNA oligonucleotide primers in the absence of ATP to assay polyadenylation. While 2-Cl-ATP resulted in primer elongation, ara-ATP and F-ara-ATP were poor substrates for yPAP. In contrast, the triphosphate derivatives of cladribine (2-Cl-dATP), clofarabine (Cl-F-ara-ATP) and its corresponding deoxyribose derivative (Cl-F-dATP) were substrates and caused chain termination in the absence of ATP. We further investigated whether analogue incorporation at the 3'-terminus of RNA primers negatively impacts polyadenylation with ATP by generating RNA oligonucleotides containing either a terminal clofarabine, Cl-F-dAdo, or cladribine residue. Incorporation of any of these analogs blocks the ability of yPAP to extend RNA past the analogue site, impeding the addition of a poly(A)-tail. To determine whether modified ATP analogues exhibit a concentration-dependent effect on polyadenylation, poly(A)-tail synthesis by yPAP with modified ATP analogues in combination with a constant level of ATP was also examined. With all the ATP analogues assayed in these studies, there was a significant reduction in poly(A)-tail length with increasing amounts of analogue triphosphate. Taken together, our results suggest that polyadenylation inhibition may be a component in the mechanism of action of adenosine analogues.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Antineoplásicos/química , Nucleótidos de Desoxiadenina/química , Poliadenilación , Polinucleotido Adenililtransferasa/antagonistas & inhibidores , Nucleótidos de Adenina/química , Arabinonucleósidos/química , Cladribina/química , Clofarabina , Nucleótidos de Desoxiadenina/metabolismo , Desoxiadenosinas/química , Polinucleotido Adenililtransferasa/metabolismo , Especificidad por SustratoRESUMEN
Parenterally administered cladribine (2-chloro-2'-deoxyadenosine) has demonstrated promising efficacy and safety in clinical trials in patients with multiple sclerosis (MS). An oral formulation of this small molecule would be an attractive option for patients. Here, we describe the chemical characterisation of the inclusion complex between cladribine and the drug carrier molecule 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD). Several techniques were used to analyse the complex both in solution and in the solid state. These analyses provided evidence that the inclusion complex cannot be simply reduced to the sum of the two species, as it shows behaviour different from that of the physical mixture of the two components. Furthermore, solution nuclear magnetic resonance spectroscopy demonstrated the existence of an inclusion complex between cladribine and 2-HP-beta-CD. Importantly, analysis of a tablet formulation demonstrated that the chemical characteristics of the inclusion complex are not affected by the manufacturing process, and that the complex is stable during storage. This tablet formulation is currently under investigation for the treatment of patients with MS.