RESUMEN
Background: Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations. Patients and methods: Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly. Results: LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein -/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved. Conclusions: These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Carcinoma Neuroendocrino/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Neoplasias de la Tiroides/tratamiento farmacológico , Adulto , Neoplasias Encefálicas/secundario , Carbazoles/farmacología , Carbazoles/uso terapéutico , Carcinoma Neuroendocrino/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Piperidinas/farmacología , Piperidinas/uso terapéutico , Prueba de Estudio Conceptual , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/genética , Pirazoles/farmacología , Piridinas/farmacología , Neoplasias de la Tiroides/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The objective of this study was to determine whether the thiol drug, diethyldithiocarbamate (DEDC) and its two metabolites, disulfiram (DS) and carbon disulfide (CS2) could be used as inhibitors of cytochrome P-450IIE1 to protect hepatocytes from cytotoxic xenobiotics. (1) Hepatocytes isolated from rats following pyrazole administration to induce cytochrome P-450IIE1 were much more susceptible to carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN) than hepatocytes from untreated rats. Microsomes isolated from P-450IIE1-induced liver were also much more effective at catalysing a NADPH-dependent metabolism of CCl4 and DMN. The activities of aniline hydroxylase and p-nitroanisole-O-demethylase increased whereas ethoxyresorufin-O-dealkylase activity was much less induced and pentoxyresorufin-O-dealkylase activity was decreased. The P-450IIE1 antibody markedly inhibited the NADPH-dependent metabolism of these compounds indicating that IIE1 is a major catalyst of the microsomal metabolism of CCl4 and DMN. (2) Hepatocytes isolated from rats treated with DEDC or its metabolites, DS and CS2, on the other hand, were resistant to CCl4 and DMN. Microsomes isolated from the liver of animals treated with DEDC or DS or CS2 were also much less effective at catalysing the NADPH-dependent metabolism of the above compounds. DEDC markedly decreased the activities of aniline hydroxylase, p-nitroanisole-O-demethylase and pentoxyresorufin-O-dealkylase but had no effect on ethoxyresorufin-O-dealkylase activity. (3) Hepatocytes isolated from pyrazole-treated rats were also more susceptible to bromobenzene (BB) and naphthalene-induced cytotoxicity than hepatocytes from untreated rats. Furthermore, DEDC or CS2 administration beforehand significantly protected hepatocytes against both xenobiotics. (4) By contrast, hepatocytes isolated from P-450IIE1 induced rats were not more susceptible to lactonitrile or cyclophosphamide. Instead, cyclophosphamide was activated by phenobarbital-induced P-450 isozymes whereas lactonitrile was activated by alcohol dehydrogenase. Hepatocytes isolated from DEDC-treated rats were also resistant to cyclophosphamide but not lactonitrile. (5) The above results suggest that P-450IIE1 catalyses the cytotoxic activation of CCl4, DMN, BB and naphthalene but not of lactonitrile or cyclophosphamide. Furthermore, the administration of DEDC and its metabolites, disulfiram or CS2, inactivates P-450IIE1 so that the hepatocytes become resistant to these hepatotoxins.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Inhibidores Enzimáticos del Citocromo P-450 , Ditiocarba/farmacología , Hepatopatías/prevención & control , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Animales , Disulfuro de Carbono/farmacología , Tetracloruro de Carbono/metabolismo , Tetracloruro de Carbono/toxicidad , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/metabolismo , Dimetilnitrosamina/metabolismo , Dimetilnitrosamina/toxicidad , Disulfiram/farmacología , Masculino , Microsomas Hepáticos/enzimología , NADP/farmacología , Oxidorreductasas N-Desmetilantes/metabolismo , Pirazoles/farmacología , Ratas , Ratas EndogámicasRESUMEN
The present study was carried out to elucidate the mechanism by which the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited an antidotal effect against acetaminophen-induced hepatotoxicity in vivo. DEDC was found to act as an antidote against acetaminophen-induced cytotoxicity in hepatocytes isolated from a pyrazole-pretreated rat without affecting cytochrome P-450 levels. The mechanism of protection exhibited against reactive intermediate N-acetyl-p-benzoquinoneimine (NAPQI)-induced cytotoxicity by DEDC was then investigated and compared with that exhibited by the permeable thiol-reductant dithiothreitol (DTT). Cytotoxicity induced by the dimethylated analogue 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI) was prevented if the hepatocytes were preincubated with DEDC for 5 min and removed before addition of 2,6-diMeNAPQI. Both DEDC and DTT were also found to act as antidotes against NAPQI- and 2,6-diMeNAPQI-induced cytotoxicity in isolated rat hepatocytes if added within 2 min of the addition of the quinoneimines. However, the addition of DEDC or DTT 10 min after either quinoneimine did not prevent subsequent cytotoxicity or restore GSH levels, indicating that the alkylation of GSH and of protein thiols was irreversible at that time. Fast atom bombardment mass spectrometry was used to show that DEDC formed conjugates with both NAPQI and 2,6-diMeNAPQI. Furthermore, these conjugates were found to be nontoxic. This suggests that DEDC acts as a trap for the toxic quinoneimines, thus preventing alkylation of essential macromolecules. In contrast, DTT reduced the quinoneimines to their respective nontoxic parent compounds and presumably also reduced mixed-protein disulfides and GSSG, thereby regenerating protein thiols and GSH. Therefore, this study suggests that DEDC and DTT act as antidotes by two different mechanisms.
Asunto(s)
Benzoquinonas/toxicidad , Ditiotreitol/farmacología , Ditiocarba/farmacología , Iminas/toxicidad , Acetaminofén/toxicidad , Alquilación/efectos de los fármacos , Animales , Benzoquinonas/metabolismo , Supervivencia Celular/efectos de los fármacos , Glutatión/metabolismo , Iminas/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Oxidación-Reducción , Ratas , Ratas EndogámicasRESUMEN
The increase in 1,4-naphthoquinone-2-sulfonate (NQS)-induced hemolysis by the superoxide dismutase inhibitor diethyldithiocarbamate (DEDC) was formerly attributed to increased superoxide anion levels in the erythrocyte. Our results show that removal of DEDC after preincubation and prior to the addition of NQS did not produce a significant increase in hemolysis, which suggests that hemolysis is primarily caused by the reaction products of DEDC with NQS and not to the inactivation of superoxide dismutase. Disulfiram, the oxidized product of DEDC, was found to be the main product formed when excess DEDC was reacted with NQS. Oxygen uptake also occurred and hydrogen peroxide was formed. The latter caused the oxidation of DEDC to disulfiram as catalase prevented disulfiram formation. Disulfiram was found to readily hemolyze erythrocytes at low concentrations as well as to crosslink the proteins in the erythrocyte membrane. Furthermore, disulfiram-induced hemolysis was markedly enhanced in glutathione-depleted erythrocytes. Disulfiram was subsequently found to readily oxidize glutathione in red blood cells. When equimolar concentrations of DEDC and NQS were reacted, the major product formed was the diethyldithiocarbamate:1,4-naphthoquinone (DEDC:NQS) conjugate. However, the principal mediator of erythrocyte hemolysis when excess DEDC is reacted with 1,4-naphthoquinone-2-sulfonate is disulfiram, whose mode of action may be to modify membrane protein sulfhydryls.
Asunto(s)
Disulfiram/farmacología , Ditiocarba/farmacología , Membrana Eritrocítica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Naftoquinonas/farmacología , Animales , Reactivos de Enlaces Cruzados , Glutatión/sangre , Humanos , Técnicas In Vitro , Masculino , Proteínas de la Membrana/metabolismo , Consumo de Oxígeno , Ratas , Ratas Endogámicas , Superóxido Dismutasa/antagonistas & inhibidoresAsunto(s)
Benzoquinonas/antagonistas & inhibidores , Ditiocarba/farmacología , Iminas/antagonistas & inhibidores , Hígado/efectos de los fármacos , Alquilación , Animales , Antídotos , Benzoquinonas/toxicidad , Supervivencia Celular/efectos de los fármacos , Glutatión/metabolismo , Iminas/toxicidad , Técnicas In Vitro , Hígado/metabolismo , Masculino , Ratas , Ratas EndogámicasRESUMEN
High doses of BHA cause hyperplasia and subsequent neoplasia in the rodent forestomach and can inhibit gastric prostaglandin (PG) synthesis in vitro. This paper examines the hypothesis that BHA induced forestomach hyperplasia occurs in response to a reduction of gastric mucus, with consequent irritation of the forestomach. This could result from inhibition of the formation of the PG's which mediate the synthesis and release of protective mucus. Groups of 10 rats received 0 or 2% BHA in the diet for 1 or 3 weeks and a positive control group was fed a diet containing indomethacin (3.5 mg/kg), a potent inhibitor of PG synthesis. After 1 week BHA caused focal erosion and ulceration of the forestomach consistent with an irritant effect, but 2 weeks later the epithelium was healed, thickened and markedly hyperplastic. Histochemical staining for mucus showed that the development of forestomach hyperplasia was associated with increased amounts of gastric and duodenal mucus and increased numbers of serotonergic-cells in the gastric and duodenal epithelium. In contrast, indomethacin caused a marked reduction in both gastric and Brunner's gland mucus. Neither BHA nor indomethacin exerted an effect on one specific type of mucus (viz: neutral, acidic or mixed) in the stomach. These results do not support the hypothesis that forestomach hyperplasia arises from an inhibition of either the synthesis or release of gastric mucus. It is possible that the increased numbers of serotonergic-cells are related to the initial ulcerative, or subsequent hyperplastic response.
Asunto(s)
Hidroxianisol Butilado/toxicidad , Inhibidores de la Ciclooxigenasa , Mucosa Gástrica/efectos de los fármacos , Indometacina/farmacología , Estómago/efectos de los fármacos , Administración Oral , Animales , Peso Corporal/efectos de los fármacos , Glándulas Duodenales/efectos de los fármacos , Glándulas Duodenales/patología , Hiperplasia/inducido químicamente , Masculino , Ratas , Ratas Endogámicas , Estómago/patologíaRESUMEN
The copper-chelating thiol drug diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced alkylation cytotoxicity by reacting with benzoquinone and forming a conjugate which was identified by fast atom bombardment mass spectrometry as 2-(diethyldithiocarbamate-S-yl) hydroquinone. In contrast to benzoquinone, the conjugate was not cytotoxic to isolated hepatocytes. The thiol reductant dithiothreitol had no effect on benzoquinone-induced alkylation cytotoxicity. However, inactivation of catalase in the hepatocytes with azide and addition of the reducing agent ascorbate markedly enhanced the cytotoxicity of the conjugate but did not affect benzoquinone-induced cytotoxicity. Furthermore, inactivation of glutathione reductase and catalase in hepatocytes greatly enhanced the cytotoxicity of the conjugate and caused oxidation of GSH to GSSG. The conjugate also stimulated cyanide-resistant respiration, which suggests that the conjugate undergoes futile redox cycling resulting in the formation of hydrogen peroxide which causes cytotoxicity in isolated hepatocytes only if the peroxide detoxifying enzymes are inactivated. Diethyldithiocarbamate does, however, protect uncompromised isolated hepatocytes from benzoquinone cytotoxicity by conjugating benzoquinone, thereby preventing the electrophile from alkylating essential macromolecules. Diethyldithiocarbamate therefore changed the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress, which was less toxic.
Asunto(s)
Benzoquinonas/farmacología , Ditiocarba/farmacología , Hígado/citología , Animales , Ácido Ascórbico/farmacología , Benzoquinonas/química , Catalasa/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ditiocarba/química , Interacciones Farmacológicas , Cinética , Hígado/efectos de los fármacos , Masculino , Oxidación-Reducción , Ratas , Ratas Endogámicas , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in a concentration-dependent fashion between 2.0-5.0 M bromotrichloromethane. 2. Lipid peroxidation (malondialdehyde) increased in a time-dependent fashion but in contrast to toxicity reached a maximum level at 2.0 mM bromotrichloromethane. 3. Hypoxia increased the toxicity of bromotrichloromethane three-fold but only decreased the amount of lipid peroxidation to a small degree. 4. In spite of this poor correlation between toxicity and lipid peroxidation, the antioxidant butylated hydroxyanisole and the iron chelator desferal protected the cells from toxicity under both aerobic and hypoxic conditions and prevented lipid peroxidation. 5. During treatment with bromotrichloromethane, cellular glutathione levels slowly decreased and oxidized glutathione appeared in the media. The addition of cystine to the incubation media prevented the formation of extracellular oxidized glutathione, indicating that cellular glutathione had leaked from the cell during treatment and was oxidized in the incubation media. Although this suggested that glutathione does not play a protective role against bromotrichloromethane toxicity, diethyl maleate-pretreatment of the cells to decrease glutathione levels markedly increased bromotrichloromethane toxicity. 6. The addition of ascorbic acid to the incubation media increased bromotrichloromethane toxicity. This was attributed to the reductive activation of bromotrichloromethane in an iron and oxygen-dependent reaction. 7. It was concluded that peroxidation of essential phospholipids contributes to bromotrichloromethane-induced hepatocyte cytotoxicity.
Asunto(s)
Bromotriclorometano/toxicidad , Supervivencia Celular/efectos de los fármacos , Hígado/citología , Aerobiosis , Anaerobiosis , Animales , Ácido Ascórbico/farmacología , Biotransformación/efectos de los fármacos , Glutatión/metabolismo , Técnicas In Vitro , Cinética , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
The copper-chelating thiol drug, diethyldithiocarbamate (DEDC) had previously been used to inhibit superoxide dismutase (SOD) and enhance oxidative stress mediated cytotoxicity. Using isolated rat hepatocytes, it was confirmed that DEDC enhances oxidative stress cytotoxicity induced by 1,4-naphthoquinone (1,4-NQ) and 1,4-naphthoquinone-2-sulphonate (1,4-NQ-2S). However, equimolar concentrations of DEDC also enhances cytotoxicity induced by benzoquinone, previously shown to cause cytotoxicity as a result of alkylation and not oxidative stress. Higher DEDC concentrations on the other hand protected against benzoquinone-induced cytotoxicity. Finally, the susceptibility of hepatocytes to quinone mediated oxidative stress cytotoxicity was not enhanced if the DEDC was removed before incubating the hepatocytes with naphthoquinone or benzoquinone. Enhanced oxidative stress cytotoxicity was only observed if the DEDC was present when hepatocytes were treated with quinones. It was concluded that DEDC forms conjugates with quinones which undergo futile redox cycling in the hepatocyte and form H2O2 as well as increase the susceptibility of hepatocytes to H2O2.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Ditiocarba/farmacología , Hígado/citología , Quinonas/toxicidad , Animales , Glutatión/metabolismo , Técnicas In Vitro , Hígado/efectos de los fármacos , Masculino , Naftoquinonas/farmacología , Oxidación-Reducción , Quinonas/metabolismo , Ratas , Ratas Endogámicas , Superóxido Dismutasa/antagonistas & inhibidoresRESUMEN
The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.