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1.
J Med Chem ; 43(25): 4850-67, 2000 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-11123995

RESUMEN

The synthesis, structure-activity relationships, and biological properties of a novel series of potent and selective phosphodiesterase type 4 (PDE4) inhibitors are described. These new aminodiazepinoindoles displayed in vitro PDE4 activity with submicromolar IC(50) values and PDE4 selectivity vs PDE1, -3, and -5. Specifically, one compound (CI-1044, 10e) provided efficient in vitro inhibition of TNFalpha release from hPBMC and hWB with IC(50) values of 0.34 and 0.84 microM, respectively. This compound was found to exhibit potent in vivo activity in antigen-induced eosinophil recruitment in Brown-Norway rats (ED(50) = 3.2 mg/kg po) and in production of TNFalpha in Wistar rats (ED(50) = 2.8 mg/kg po). No emetic side effects at therapeutic doses were observed in ferrets.


Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Antiasmáticos/síntesis química , Antiinflamatorios no Esteroideos/síntesis química , Azepinas/síntesis química , Indoles/síntesis química , Niacinamida/síntesis química , Inhibidores de Fosfodiesterasa/síntesis química , 3',5'-GMP Cíclico Fosfodiesterasas , Animales , Antiasmáticos/efectos adversos , Antiasmáticos/química , Antiasmáticos/farmacología , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Aorta/enzimología , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacología , Unión Competitiva , Encéfalo/metabolismo , Lavado Broncoalveolar , Línea Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Perros , Eosinófilos/patología , Hurones , Cobayas , Humanos , Técnicas In Vitro , Indoles/efectos adversos , Indoles/química , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Masculino , Monocitos/enzimología , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacología , Ovalbúmina/inmunología , Fosfodiesterasa I , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Relación Estructura-Actividad , Tráquea/enzimología , Factor de Necrosis Tumoral alfa/biosíntesis , Vómitos/inducido químicamente
2.
Methods Find Exp Clin Pharmacol ; 21(2): 99-104, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10327390

RESUMEN

Expression of the COX-2 enzyme has been reported in animal models of inflammatory bowel disease (IBD) as well as in patients affected by ulcerative colitis and Crohn's disease. Recently, selective inhibitors of COX-2 have become available. In this study we have evaluated three highly selective COX-2 inhibitors, NS-398, SC-58125 and PD-138387, on the trinitro-benzene sulfonic acid (TNBS) model of colitis in rats. Daily oral administration of the three compounds evaluated up to a dose of 100 mg/kg failed to significantly modify any of the parameters evaluated. Our data show that despite their potent extraintestinal antiinflammatory activity, COX-2 inhibitors do not seem to have any beneficial effect in TNBS colitis and raise the question whether this therapeutic approach would be beneficial in patients with IBD.


Asunto(s)
Colitis/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Isoenzimas/efectos de los fármacos , Peroxidasas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Colitis/inducido químicamente , Colitis/enzimología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/antagonistas & inhibidores , Masculino , Nitrobencenos/uso terapéutico , Peroxidasa/antagonistas & inhibidores , Fenoles/uso terapéutico , Pirazoles/uso terapéutico , Ratas , Ratas Sprague-Dawley , Sulfonamidas/uso terapéutico , Tiazoles/uso terapéutico , Ácido Trinitrobencenosulfónico
3.
J Med Chem ; 42(7): 1151-60, 1999 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-10197959

RESUMEN

Selective cyclooxygenase-2 (COX-2) inhibitors have been shown to be potent antiinflammatory agents with fewer side effects than currently marketed nonsteroidal antiinflammatory drugs (NSAIDs). Initial mass screening and subsequent structure-activity relationship (SAR) studies have identified 4b (PD138387) as the most potent and selective COX-2 inhibitor within the thiazolone and oxazolone series of di-tert-butylphenols. Compound 4b has an IC50 of 1.7 microM against recombinant human COX-2 and inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.17 microM. It was inactive against purified ovine COX-1 at 100 microM and did not inhibit COX-1 activity in platelets at 20 microM. Compound 4b was also orally active in vivo with an ED40 of 16 mg/kg in the carrageenan footpad edema (CFE) assay and caused no gastrointestinal (GI) damage in rats at the dose of 100 mg/kg but inhibited gastric prostaglandin E2 (PGE2) production in rats' gastric mucosa by 33% following a dose of 100 mg/kg. The SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes. A simple isosteric replacement led to the reversal of selectivity.


Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Isoenzimas/metabolismo , Oxazoles/síntesis química , Fenoles/síntesis química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiazoles/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/toxicidad , Carragenina/toxicidad , Línea Celular , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/toxicidad , Dinoprostona/antagonistas & inhibidores , Edema/inducido químicamente , Edema/tratamiento farmacológico , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Masculino , Proteínas de la Membrana , Ratones , Oxazoles/química , Oxazoles/farmacología , Oxazoles/toxicidad , Fenoles/química , Fenoles/farmacología , Fenoles/toxicidad , Ratas , Ratas Sprague-Dawley , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patología , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Tiazoles/toxicidad
4.
J Med Chem ; 42(7): 1161-9, 1999 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-10197960

RESUMEN

Two isoforms of the cyclooxygenase (COX) enzyme have been identified: COX-1, which is expressed constitutively, and COX-2, which is induced in inflammation. Recently, it has been shown that selective COX-2 inhibitors have antiinflammatory activity and lack the GI side effects typically associated with NSAIDs. Initial mass screening and subsequent SAR studies have identified 6b (PD164387) as a potent, selective, and orally active COX-2 inhibitor. It had IC50 values of 0.14 and 100 microM against recombinant human COX-2 and purified ovine COX-1, respectively. It inhibited COX-2 activity in the J774A.1 cell line with an IC50 of 0.18 microM and inhibited COX-1 activity in platelets with an IC50 of 3.1 microM. The choline salt of compound 6b was also orally active in vivo with an ED40 of 7. 1 mg/kg in the carrageenan footpad edema (CFE) assay. In vivo studies in rats at a dose of 100 mg/kg showed that this compound inhibited gastric prostaglandin E2 (PGE2) production in gastric mucosa by 77% but caused minimal GI damage. SAR studies of this chemical series revealed that the potency and selectivity are very sensitive to minor structural changes.


Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Isoenzimas/metabolismo , Fenoles/síntesis química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tiadiazoles/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/toxicidad , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Carragenina/toxicidad , Línea Celular , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/toxicidad , Dinoprostona/antagonistas & inhibidores , Edema/inducido químicamente , Edema/tratamiento farmacológico , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Humanos , Hiperalgesia/tratamiento farmacológico , Técnicas In Vitro , Masculino , Proteínas de la Membrana , Ratones , Fenoles/química , Fenoles/farmacología , Fenoles/toxicidad , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Ovinos , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patología , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacología , Tiadiazoles/toxicidad
5.
Exp Cell Res ; 244(1): 302-9, 1998 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-9770373

RESUMEN

The caspase family of proteases has previously been implicated in the biochemical cascade leading to apoptotic cell death. Recently caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following phytohemagglutinin (PHA) activation of peripheral blood mononuclear cells (C. Miossec et al., J. Biol. Chem. 272, 13459-13462). More recently, J. M. Zapata and colleagues (J. Biol. Chem. 273, 6916-6920, 1998), however, proposed that caspase-3 activity detected during T-cell activation was due to a methodological artifact related to the composition of the cell lysis buffer. Here we show that in PHA-activated Jurkat T-cells using the recommended lysis buffer detailed by Zapata et al., a caspase-3-like protease is activated and is accompanied by cleavage of PARP and alpha-spectrin into cleavage products suggestive of caspase-3 proteolytic activation. LDH release did not increase following PHA stimulation in this paradigm. Two caspase inhibitors, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) and acetyl-Asp-Glu-Val-Asp-CHO, blocked IL-2 release in a dose-dependent manner. Caspase-3-like protease-generated PARP and alpha-spectrin breakdown product formation was also reduced by Z-D-DCB. In addition, Jurkat T-cells costimulated with anti-CD3 plus anti-CD28 produced significant levels of IL-2 that were also blocked by these caspase inhibitors. Importantly, IL-2 was determined in cell culture supernatants, thus avoiding a cell lysis step that might have enabled activation of caspase-3 by granzyme B. Collectively, these data support the role of caspase-3-like protease activity in Jurkat T-cell activation and demonstrate that caspase-3 like activity is necessary for IL-2 release in PHA-activated and anti-CD3/anti-CD28 costimulated Jurkat T-cells.


Asunto(s)
Caspasas/metabolismo , Caspasas/fisiología , Interleucina-2/metabolismo , Activación de Linfocitos , Linfocitos T/enzimología , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Caspasa 3 , Inhibidores de Caspasas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiología , Humanos , Hidrólisis , Interleucina-2/antagonistas & inhibidores , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Oligopéptidos/farmacología , Péptido Hidrolasas/metabolismo , Fitohemaglutininas/farmacología , Linfocitos T/inmunología
6.
J Pharmacol Exp Ther ; 287(1): 301-6, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9765350

RESUMEN

Nonsteroidal anti-inflammatory drugs often cause development of significant GI lesions. Selective inhibitors of prostaglandin G/H synthase/cyclooxygenase-2 (PGHS-2) enzyme and some dual inhibitors of PGHS/5-lipoxygenase (5-LO) enzymes have been reported to be potent anti-inflammatory compounds that carry a much lower risk of having GI irritating effects. We have evaluated the anti-inflammatory effect and the GI safety profile of three new anti-inflammatory compounds: the selective PGHS-2 inhibitors NS-398 and PD 138387 and the PGHS/5-LO dual inhibitor PD 137968. All the compounds tested showed an anti-inflammatory activity in the carragenan footpad edema test in rats. None of these compounds caused either gastric damage 4 h after p.o. administration of 100 mg/kg in rats or inhibition of PGE2 synthesis in the stomach. However, when administered p.o. at an effective anti-inflammatory dose to rats with pre-existing acetic acid-induced gastric ulcer, NS-398 caused a statistically significant delay of ulcer healing. No impairment of the ulcer healing was observed with the other compounds evaluated. Derivatives of 2,6-di-tert-butylphenol, whose members may act as PGHS-1/PGHS-2 inhibitors, selective PGHS-2 inhibitors or PGHS/5-LO dual inhibitors, are novel anti-inflammatory compounds that are devoid of GI irritating effects and do not affect the rate of pre-existing gastric ulcer healing.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/efectos de los fármacos , Inhibidores de la Lipooxigenasa , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Úlcera Gástrica/fisiopatología , Ácido Acético , Animales , Antiinflamatorios no Esteroideos/toxicidad , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Mucosa Gástrica/efectos de los fármacos , Indometacina/farmacología , Masculino , Proteínas de la Membrana , Nitrobencenos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sulfonamidas/farmacología
7.
Immunopharmacology ; 37(2-3): 231-44, 1997 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9403342

RESUMEN

Children with congenital homozygous deficiency of purine nucleoside phosphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cell death has been attributed to intracellular accumulation of dGTP, a potent inhibitor of ribonucleotide reductase and subsequently DNA synthesis, in thymocytes and T-cells but not B-cells. However, the mode of cell death has not been determined to be either necrosis or apoptosis. To examine the involvement of apoptosis in T-cells following PNP inhibition, MOLT-4 cells, a human T cell leukemia cell line, were co-treated with the PNP inhibitor, CI-1000 (2-amino 3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resulted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([3H]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33,258 showed nuclear morphology characteristic of apoptosis. Western blots (24 h lysates) were probed with antibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-alpha-spectrin revealed the accumulation a 120 kD breakdown product, both suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine protease). Western blots also detected the loss of the intact 32 kD caspase-3 isoform, a biochemical event associated with caspase-3 activation. Corresponding fluorometric activity assays detected a marked increase in caspase-3-like activity using the substrate Ac-DEVD-MCA. Lastly, a pan caspase inhibitor (Z-D-DCB) and 2'-deoxycytidine (dCyd), which is known to prevent dGTP accumulation following PNP inhibition, were able to prevent cell death and all indicators of caspase-3-like activity in MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provided several lines of evidence for the role of apoptosis and the contribution of caspase-3-like proteases in T-cell death following PNP inhibition.


Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Linfocitos T/citología , Linfocitos T/enzimología , Apoptosis/fisiología , Caspasa 3 , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Cisteína Endopeptidasas/efectos de los fármacos , ADN/efectos de los fármacos , ADN/metabolismo , Desoxiguanosina/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas/metabolismo , Espectrina/metabolismo , Linfocitos T/efectos de los fármacos , Timidina/farmacocinética
10.
J Neurochem ; 68(6): 2328-37, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9166725

RESUMEN

We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.


Asunto(s)
Apoptosis/fisiología , Caspasas , Cisteína Endopeptidasas/metabolismo , Especificidad de Anticuerpos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacología , Caspasa 1 , Caspasa 3 , Cumarinas , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/inmunología , ADN/efectos de los fármacos , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/análisis , Precursores Enzimáticos/inmunología , Precursores Enzimáticos/metabolismo , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Humanos , Hidrólisis , Neuroblastoma , Neuronas/citología , Neuronas/enzimología , Oligopéptidos , Poli(ADP-Ribosa) Polimerasas/análisis , Poli(ADP-Ribosa) Polimerasas/inmunología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteasas/farmacología , Estaurosporina/farmacología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/enzimología
11.
Biochem J ; 319 ( Pt 3): 683-90, 1996 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-8920967

RESUMEN

The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.


Asunto(s)
Apoptosis , Calpaína/metabolismo , Cisteína Endopeptidasas/metabolismo , Neuronas/fisiología , Oxocinas , Espectrina/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 1 , Línea Celular , Células Cultivadas , Cerebelo/citología , Cerebelo/enzimología , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Cinética , Toxinas Marinas/farmacología , Datos de Secuencia Molecular , Neuroblastoma , Neuronas/citología , Neuronas/enzimología , Células PC12 , Ratas , Estaurosporina/farmacología , Especificidad por Sustrato , Células Tumorales Cultivadas
12.
Scand J Immunol ; 39(3): 327-32, 1994 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8128192

RESUMEN

Patients with homozygous deficiency of purine nucleoside phosphorylase (PNP) present with a T-cell selective immune deficiency. To elucidate the potential use of PNP inhibitors in the therapy of cutaneous T-cell lymphomas (CTCLs) the authors studied the effects of CI-1000 (formerly PD141955-2) and CI-972 on a T-cell line MyLa established from a patient with mycosis fungoides. Both PNP inhibitors had significant, dose-dependent, inhibitory effects on the proliferation of the T-cell line. CI-1000 (ED50: 3.7 microM) was approximately six-fold more potent in blocking 3H-thymidine uptake than CI-972 (ED50: 22.5 microM). The inhibitory effect of either substance could not be increased by addition of deoxyguanosine. Flow cytometric analysis revealed that both PNP inhibitors caused a block in the S-phase of the cell cycle. The inhibitory effect on proliferation was reversible partially by addition of IL-2. When testing proliferation inhibition of both substances on an IL-2-dependent T-cell line (SeAx), their inhibitory effects were reduced significantly. These data document a mechanism of action of the PNP inhibitors independent of deoxyguanosine and partially reversible by IL-2. The authors' observations suggest the potential use of PNP inhibitors in the therapy of cutaneous T-cell lymphomas and provide evidence for a pathway independent from deoxyguanosine by which PNP inhibitors might function in T cells.


Asunto(s)
Desoxiguanosina/farmacología , Interleucina-2/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Linfocitos T/inmunología , Ciclo Celular , Línea Celular , Citometría de Flujo , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Interleucina-7/farmacología , Activación de Linfocitos , Micosis Fungoide , Pirimidinas/farmacología , Fase S , Síndrome de Sézary , Tiofenos/farmacología , Factores de Tiempo
14.
Adv Exp Med Biol ; 370: 167-71, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-7660883

RESUMEN

Purine nucleosides HxR or GdR (2.5 micrograms/mL blood) were added to EDTA-treated cynomolgus monkey whole blood in vitro, alone or with the PNP inhibitor CI-1000 (1 microgram/mL), mixed, and the concentration of nucleosides remaining in plasma followed as a function of time. The half-lives of GdR and HxR in control blood were 1.2 and < 1 min, respectively, and were extended to 17.8 and 39.8 min, respectively, by coaddition of CI-1000. In contrast, a structural analog of CI-1000, CI-972, when tested in parallel at 1 microgram/mL, had markedly less effect on the breakdown of either nucleoside. The ability of CI-1000 to retard nucleoside breakdown in blood in vitro may be a predictor of in vivo activity, and can be viewed as an early and essential biochemical consequence of PNP inhibition culminating in immunosuppression.


Asunto(s)
Desoxiguanosina/sangre , Guanina/análogos & derivados , Inosina/sangre , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Animales , Ácido Edético , Guanina/farmacología , Semivida , Humanos , Cinética , Macaca fascicularis , Ratas
16.
Agents Actions ; 39 Spec No: C96-8, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8273599

RESUMEN

Inhibitors of purine nucleoside phosphorylase (PNP) are of interest as potential T-cell-selective immunosuppressive agents and for other uses. PD 141955 (9-deaza-9-(3-thienylmethyl)guanine; 2-amino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrimidin -4-one) is 12- to 100-fold more potent than CI-972 (8-amino-9-deaza-9-(3-thienylmethyl)guanine; 2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrim idin-4- one) in PNP enzyme inhibition assays. In the human MLR, PD 141955 has IC50s of 2.8 and 12.8 microM in the presence and absence, respectively, of 15 microM GdR (means from 10 assays), while the IC50s of CI-972 tested in parallel are > 30 microM. Concentration-dependent accumulation of dGTP occurs in PD 141955-treated MLRs under conditions in which CI-972 lacks detectable activity. Thus, consistent with its greater PNP inhibitory activity in a cell free system, PD 141955 is significantly more potent than CI-972 in its ability to suppress the MLR.


Asunto(s)
Guanina/análogos & derivados , Inmunosupresores/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinas/farmacología , Linfocitos T/inmunología , Tiofenos/farmacología , Células Cultivadas , Guanina/farmacología , Humanos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos T/efectos de los fármacos
17.
Agents Actions ; 39 Spec No: C99-101, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8273600

RESUMEN

Patients with deficiency in purine nucleoside phosphorylase (PNP) have elevated levels of the PNP substrates inosine, guanosine, and (rarely) 2'-deoxyguanosine (GdR) in their plasma and urine. GdR is critical because it serves as a precursor of dGTP, which blocks T-cell replication, thus leading to T-cell-selective immune dysfunction. We adapted these findings to the study of PNP inhibitors in human and rat blood in vitro. Blood was spiked with GdR (2.5 micrograms/ml) and the effects of PD 141955 (9-deaza-9-(3-thienylmethyl)guanine; 2-amino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrimidin -4-one) and CI-972 (8-amino-9-deaza-9-(3-thienylmethyl)guanine; 2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrim idin-4- one) on GdR catabolism were determined. GdR was metabolized 89 times faster in human blood than in rat blood (half-life = 12.0 +/- 1.4 s in human blood). When PD 141955 (1 microgram/ml) was added to human blood before spiking, the GdR half-life increased to > 60 min. In contrast, CI-972 (1 microgram/ml) extended the GdR half-life to 7.2 +/- 1.7 min. Both PD 141955 and CI-972 at 1 microgram/ml significantly retarded GdR catabolism from rat blood.


Asunto(s)
Desoxiguanosina/sangre , Guanina/análogos & derivados , Inmunosupresores/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinas/farmacología , Tiofenos/farmacología , Animales , Cromatografía Líquida de Alta Presión , Desoxiguanosina/orina , Guanina/farmacología , Semivida , Humanos , Ratas
18.
Biochem Pharmacol ; 44(5): 996-9, 1992 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-1530667

RESUMEN

An in-parallel comparison is presented of the in vitro and in vivo properties of two 9-deazaguanine analog inhibitors of purine nucleoside phosphorylase (PNP), CI-972 [8-amino-9-deaza-9-(3-thienylmethyl)guanine] and PD 141955 [9-deaza-9-(3-thienylmethyl)guanine] (published Ki values of 0.83-8.0 and 0.08 microM, respectively). Despite structural similarities, PD 141955 was considerably more potent and active in all systems studied. The respective IC50 values for inhibition of MOLT-4 cell growth in the absence and presence of 10 microM 2'-deoxyguanosine (GdR) were greater than 50 and 5.06 microM for CI-972 and 15.4 and 0.061 microM for PD 141955. PD 141955 induced accumulation of dGTP in GdR-treated MOLT-4 and CEM cells at log-lower concentrations than were required of CI-972, and the magnitude of dGTP accumulation in PD 141955-treated T cell cultures was markedly greater (e.g. 366 vs 100 pmol/10(6) CEM cells at 10 microM). PD 141955 administered orally produced a dose-dependent elevation of plasma inosine and guanosine in rats over a broad concentration range. Mean plasma inosine concentrations following a 150 mg/kg p.o. dose peaked at 6.21 and 13.2 microM in CI-972 and PD 141955-treated rats, respectively. Low levels of inosine were detectable at 50 micrograms/kg following oral administration of PD 141955.


Asunto(s)
Guanina/análogos & derivados , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinas/farmacología , Tiofenos/farmacología , Animales , Línea Celular , Nucleótidos de Desoxiguanina/análisis , Relación Dosis-Respuesta a Droga , Guanina/química , Guanina/farmacología , Guanosina/sangre , Humanos , Inosina/sangre , Masculino , Pirimidinas/química , Ratas , Ratas Endogámicas , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Tiofenos/química
19.
J Med Chem ; 35(9): 1605-9, 1992 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-1578487

RESUMEN

Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme in the purine cascade and has been a target for drug design for sometime. A series of potent human PNP inhibitors, pyrrolo[3,2-d]pyrimidines (9-deazaguanines), has been synthesized and evaluated in the enzyme assay and in the cell line assay using MOLT-4 (T-cell) and MGL-8 (B-cell) lymphoblasts for selectivity. One of the compounds, 2,6-diamino-3,5- dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrimidine-4-one (11c; CI-972), was found to be moderately potent, competitive, and reversible inhibitor of PNP with Ki = 0.83 microM. It was also found to be selectively cytotoxic to MOLT-4 lymphoblasts (IC50 = 3.0 microM) but not to MGL-8 lymphoblasts and was evaluated further. Compound 11c (CI-972) is under development in the clinic.


Asunto(s)
Inmunosupresores/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinas/farmacología , Linfocitos T/efectos de los fármacos , Tiofenos/farmacología , Linfocitos B/efectos de los fármacos , Eritrocitos/enzimología , Humanos , Inmunosupresores/síntesis química , Cinética , Pirimidinas/síntesis química , Tiofenos/síntesis química
20.
J Med Chem ; 35(8): 1451-7, 1992 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-1573639

RESUMEN

A series of 3-substituted hypoxanthines (6-10, 14-17) and related analogues (22, 23) have been synthesized as inhibitors of purine nucleoside phosphorylase (PNP), which may conceivably act as T-cell-selective immunosuppressive agents with potential utility in autoimmune disorders such as rheumatoid arthritis, in organ transplantations, and in T-cell leukemias. The compounds were evaluated for their PNP activity by a radiochemical assay and also for their cytotoxic effects on a T-lymphoblastoid cell line (MOLT-4). Appropriate substitutions on 3-benzylhypoxanthine (7a) (IC50 in PNP assay, 112 microM; IC50 in MOLT-4 assay, 204.2 microM) increase potency: 8-amino (17a; 42.6, 65.2), 2-hydroxy (9a; 13.4, 28.6), 2-amino (10a; 11.4, 29.1), and 2,8-diamino (16a; 5.0, 11.9). Variation of the 3-aryl substituents of 16a as in 16b-d has thus far failed to further increase potency. Replacement of the 6-oxygen function in 7a with the analoguous nitrogen or sulfur functions, as in 22a and 23a, resulted in little change in activity. Other variations including the increase of the 3-aliphatic chain length as in 6h and 7h (n = 2), the substitution of the phenyl ring with electron-withdrawing groups as in 7e-g, and replacement of the 2-hydrogen with methylthio as in 8a and 14a resulted in decrease of activity. The values for 16a-d represent moderate but significant activities, as compared to the most active inhibitor presently known, 8-amino-9-thienylguanine (1c; 0.17, 0.82). 2,8-Diamino-3-substituted hypoxanthines (16a-d) represent a novel structural type hitherto unreported in the literature, and efficient methodologies for their synthesis were developed in the present studies. The formation of the aminoimidazole moiety occurred through a base-catalyzed 1,5-(O----N)-carbamimidoyl rearrangement (13 to 14, 20 to 16).


Asunto(s)
Hipoxantinas/síntesis química , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Línea Celular , Inhibidores Enzimáticos/síntesis química , Humanos , Hipoxantinas/farmacología , Inmunosupresores/síntesis química , Relación Estructura-Actividad
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