RESUMEN
Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous "normal" cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Pruebas Inmunológicas de Citotoxicidad/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular , Técnicas de Cocultivo , Pruebas Inmunológicas de Citotoxicidad/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Macrófagos , Ratones , Monocitos , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Cultivo Primario de Células/instrumentación , Cultivo Primario de Células/métodosRESUMEN
Toll-like receptors (TLR) are critical mediators of the immune system with their activation linked to infection, inflammation and the pathogenesis of immune diseases including autoimmunity and cancer. For this reason, over the last 2 decades, TLR and their associated signalling pathways have been targeted therapeutically to enhance innate and adaptive immunity. Several TLR ligands, both endogenous and synthetic are at various phases of clinical testing, and new ligands are continually emerging. Agonists of TLR7 are known immune response modifiers, simultaneously stimulating several cell types, resulting in immune cell activation and cytokine and chemokine release. The immune stimulating properties of the TLR7 agonist Imiquimod has also been exploited for use in the treatment of malignant superficial tumours of the skin. Here, we investigated a novel TLR7 agonist UC-1V150 and demonstrate it activates both human and mouse myeloid cells in vitro and in vivo, to deliver potent FcγR-mediated engulfment of opsonized target cells.
Asunto(s)
Benzaldehídos/química , Benzaldehídos/farmacología , Factores Inmunológicos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Glicoproteínas de Membrana/agonistas , Fagocitosis/inmunología , Purinas/química , Purinas/farmacología , Receptor Toll-Like 7/agonistas , Aminoquinolinas/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Humanos , Imiquimod , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Receptores de IgG/biosíntesisRESUMEN
Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.
Asunto(s)
Imidazoles/farmacología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/metabolismo , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Antígenos/inmunología , División Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratas , Ratas Endogámicas Lew , Staphylococcus/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Sterile perforated polyethylene spheres (wiffle golf balls) were implanted s.c. in beagle dogs. A local inflammatory reaction was elicited within the spheres by injecting carrageenan. Changes in leukocyte count, prostaglandin E2, thromboxane B2 and leukotriene B4 levels were monitored in fluid samples collected over a 24-hr period. Blood samples were also collected at various time points and analyzed for prostaglandin E2 and leukotriene B4 production after ex vivo calcium ionophore treatment. Effects of standard antiinflammatory agents (aspirin, indomethacin, dexamethasone, tenidap and zileuton) and newer cyclooxygenase-2 (COX-2) selective agents (nimesulide, nabumetone and SC-58125) were determined after oral administration. Ex vivo inhibition of cyclooxygenase product synthesis (prostaglandin E2, thromboxane B2) in whole blood was used as an indicator of activity for the constitutive COX-1 isoform, although inhibition of the synthesis of these mediators in the chamber exudate during an inflammatory process is believed to represent COX-2 inhibition. Treatment effects on leukotriene B4 production were also determined both ex vivo in whole blood and in the fluid. All of the compounds tested, except aspirin, inhibited leukocyte infiltration into the fluid exudate. Inhibitors that exert their effects on both isozymes of cyclooxygenase attenuate production of cyclooxygenase metabolites in both the inflammatory exudate and in peripheral blood ex vivo, although COX-2 selective inhibitors only demonstrated activity in the exudate. A 5-lipoxygenase inhibitor (zileuton), a corticosteroid (dexamethasone) and a dual COX-2 selective/5-lipoxygenase inhibitor (RWJ 63556) had similar profiles in that they all inhibited cell infiltration and eicosanoid production in the fluid and also attenuated leukotriene B4 production in both the fluid and blood.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Inflamación/tratamiento farmacológico , Inhibidores de la Lipooxigenasa , Inhibidores de la Lipooxigenasa/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Carragenina/toxicidad , Inhibidores de la Ciclooxigenasa/uso terapéutico , Modelos Animales de Enfermedad , Perros , Estudios de Evaluación como Asunto , Femenino , Inflamación/inducido químicamente , Leucotrieno B4/sangre , Inhibidores de la Lipooxigenasa/uso terapéutico , Masculino , Sulfonamidas/uso terapéutico , Tiofenos/uso terapéuticoRESUMEN
Compound 4k N-[5-(4-fluoro)phenoxythien-2-yl]methanesulfonamide is representative of a new class of potent inhibitors of 5-lipoxygenase (5-LO). These versatile compounds exhibit dose-dependent inhibition of 5-LO with IC50s ranging from 20-100 nM in the rat basophilic leukemia (RBL-1) cell homogenate assay and submicromolar IC50s in both the RBL-1 and human peripheral blood leukocyte (PBL) whole cell assays. Compound 4k also showed significant anti-inflammatory activity in the adjuvant arthritic rat at an oral dose of 3 mg/kg.
Asunto(s)
Inhibidores de la Lipooxigenasa/síntesis química , Sulfonamidas/química , Administración Oral , Alquilación , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Éteres/síntesis química , Humanos , Leucemia Basofílica Aguda/enzimología , Leucemia Basofílica Aguda/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Inhibidores de la Lipooxigenasa/química , Inhibidores de la Lipooxigenasa/farmacología , Inhibidores de la Lipooxigenasa/uso terapéutico , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfuros/síntesis química , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Tiofenos/química , Células Tumorales CultivadasRESUMEN
A series of nitroarylhydroxymethylphosphonic acids was synthesized and evaluated as inhibitors of CD45. It was discovered that both the alpha hydroxy and nitro groups are essential for activity. Potency is enhanced by the addition of a large lipophilic group on the aryl ring adjacent to the phosphonic acid moiety. Kinetics studies have shown that these compounds are competitive inhibitors and thus bind at the active site of this enzyme.
Asunto(s)
Antígenos Comunes de Leucocito/metabolismo , Nitrobencenos/síntesis química , Organofosfonatos/síntesis química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Modelos Químicos , Datos de Secuencia Molecular , Nitrobencenos/farmacología , Organofosfonatos/farmacología , Proteínas Recombinantes/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , SpodopteraRESUMEN
Further investigation of the structural requirements of a series of benzylphosphonic acid inhibitors of human prostatic acid phosphatase has led to the highly potent series of alpha-aminobenzylphosphonic acids. The alpha-benzylaminobenzylphosphonic acid, with an IC50 = 4 nM, exhibited a 3500-fold improvement in potency over the carbon analogue, alpha-phenylethyl. The enhanced potency may be due to a combination of four favorable interactions including those with the phosphate binding region, the presence the hydrophobic moieties of the benzylamino and phenylphosphonic acid, and a rigid conformer produced by an internal salt bridge between the phosphonate and the alpha-amino group. Replacement of the phosphonic acid moiety with a phosphinic or carboxylic acid as well as deletion of the benzyl substitution of the alpha-amino group led to great reductions in potency.
Asunto(s)
Fosfatasa Ácida/antagonistas & inhibidores , Alcoholes Bencílicos , Inhibidores Enzimáticos/síntesis química , Organofosfonatos/química , Próstata/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Masculino , Modelos Moleculares , Conformación ProteicaRESUMEN
The recently synthesized carbocyclic 2',3'-didehydro-2',3'-dideoxy-6-deoxy-6-amino-guanosine [(-)6AC] was evaluated as a prodrug for carbovir, carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine [(-)CBV] in seven male Sprague-Dawley rats. A randomized three-way cross-over design was used. Rats were assigned to receive the following treatments: a 20 mg/kg (-)6AC infusion, 40 mg/kg (-)6AC orally, and a 20 mg/kg (-)CBV infusion. Blood samples were collected over 480 min, and urine was collected for up to 48 hr. A 2- to 3-day washout period was observed between treatments. Following i.v. infusion, (-)6AC concentrations in the blood declined rapidly in a monoexponential pattern with an elimination half-life of 11.3 +/- 3.3 min (mean +/- SD, n = 7). The time-averaged total body clearance was 115.7 +/- 32.6 ml/min/kg. The fraction of the dose excreted unchanged in urine was 0.28 +/- 0.06. The fraction of the (-)6AC dose metabolized to (-)CBV was 0.48 +/- 0.14. Following oral administration of (-)6AC, the bioavailability of (-)CBV was 46.2 +/- 9.9% (n = 6) in comparison with the bioavailability of approximately 20% previously obtained after an oral dose of (-)CBV. The Cmax of (-)CBV after a 40 mg/kg oral dose of (-)6AC was 1.65 +/- 0.7 micrograms/ml as compared with the previously reported Cmax of 1.00 microgram/ml obtained after a 60 mg/kg oral dose of (-)CBV. (-)6AC has considerable potential for the improvement of the extent of absorption of (-)CBV from oral dosing.
Asunto(s)
Antivirales/farmacocinética , Didesoxinucleósidos/farmacocinética , Profármacos/farmacocinética , Animales , Antivirales/sangre , Antivirales/orina , Disponibilidad Biológica , Didesoxinucleósidos/sangre , Didesoxinucleósidos/orina , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas EndogámicasRESUMEN
A series of C-4 hydroxylated and halogenated anilino derivatives of epipodophyllotoxin or 4'-demethylepipodophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 11-17 and 22 are more potent than etoposide in causing DNA breakage, while compounds 11-13, 15, 16, and 20 are as active or more active than etoposide in their inhibition of the human DNA topoisomerase II. The cytotoxicity in KB cells appears to have no direct correlation with their ability to inhibit DNA topoisomerase II and to cause protein-linked DNA breaks in cells.
Asunto(s)
Antineoplásicos/síntesis química , Podofilotoxina/análogos & derivados , Inhibidores de Topoisomerasa II , Células Cultivadas , Fenómenos Químicos , Química , Humanos , Podofilotoxina/síntesis química , Podofilotoxina/farmacología , Relación Estructura-ActividadRESUMEN
A series of analogues of etoposide, the C-4 amino- and alkylamino-substituted 4'-demethyl-epipodophyllotoxins, have been synthesized and studied for their activity to inhibit type II human DNA topoisomerase as well as their activity in causing cellular protein-linked DNA breakage. Substitution of the glycosidic moiety of 1 by a 2"-hydroxyethylamino or 2"-methoxyethylamino chain at the C-4 beta position resulted in potent inhibitors of the human DNA topoisomerase II. This inhibitory activity correlates reasonably well with their activity in causing protein-linked DNA breakage in KB cells. The in vitro cytotoxicity (KB) appears to have no correlation with the inhibitory activity of the human DNA topoisomerase II.
Asunto(s)
Antineoplásicos/síntesis química , Podofilotoxina/análogos & derivados , Inhibidores de Topoisomerasa II , Células Cultivadas , ADN/efectos de los fármacos , Daño del ADN , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Análisis Espectral , Relación Estructura-ActividadRESUMEN
Several ring C aromatized analogues of podophyllotoxin were synthesized for testing against human DNA topoisomerase II. The results indicate that aromatization of ring C gave rise to no inhibition of this enzyme at 200 microM. A comparison of the cytotoxicity among these compounds also demonstrates that a free hydroxyl group at C-4 contributes to significant cytotoxicity.