RESUMEN
BACKGROUND AND PURPOSE: The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model. EXPERIMENTAL APPROACH: Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg kg(-1) day(-1); dalcetrapib 100, 300 or 500 mg(-1) kg day(-1); or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days' treatment with torcetrapib 40 mg kg(-1) day(-1), dalcetrapib 500 mg kg(-1) day(-1) or vehicle was measured by quantitative polymerase chain reaction. KEY RESULTS: Torcetrapib transiently increased mean AP in normotensive rats (+3.7 +/- 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 +/- 0.6 mmHg with 40 mg kg(-1) day(-1) at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas. CONCLUSIONS AND IMPLICATIONS: In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Quinolinas/toxicidad , Sistema Renina-Angiotensina/efectos de los fármacos , Compuestos de Sulfhidrilo/toxicidad , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Amidas , Animales , Anticolesterolemiantes/administración & dosificación , Anticolesterolemiantes/toxicidad , Aorta/efectos de los fármacos , Aorta/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Ésteres , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Masculino , Reacción en Cadena de la Polimerasa , Quinolinas/administración & dosificación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Sistema Renina-Angiotensina/genética , Compuestos de Sulfhidrilo/administración & dosificaciónRESUMEN
The liver X> or = receptor alpha (LXRalpha) is a nuclear receptor with a key role in bile acid biosynthesis and cholesterol metabolism. The present study investigated the expression and function of LXRalpha in the normal and malignant human breast. LXRalpha mRNA transcripts were detected by RT-PCR in nine breast carcinoma cell lines. The nucleotide sequence of the cloned PCR product was identical to the corresponding human LXRalpha cDNA sequence. Expression of LXRalpha protein was confirmed by immunoblot analysis of breast cancer cell lysates. LXRalpha mRNA was expressed in 14/15 (93%) of normal human breast mammoplasty specimens and in 11/15 (73%) of primary breast carcinomas. Oxysterol and nonsteroidal LXRalpha agonists at low micromolar concentrations inhibited proliferation of breast carcinoma cell lines in culture. The importance of LXRalpha signaling in cholesterol homeostasis and the observed expression of LXRalpha in normal breast tissue suggest that this nuclear oxysterol receptor has an important physiological function in the breast. LXRalpha gene expression is regulated by dietary fatty acids implicated in breast carcinogenesis and detection of LXRalpha expression in breast cancer cell lines and breast tumors in the present study indicates that LXRalpha may also be important in breast carcinogenesis. Inhibition of breast cancer cell proliferation suggests that pharmacological LXRalpha agonists may have potential preventive and/or therapeutic antitumor activity in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma/genética , Carcinoma/patología , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/biosíntesis , Mama/patología , Colesterol/metabolismo , Proteínas de Unión al ADN , Femenino , Humanos , Immunoblotting , Receptores X del Hígado , Receptores Nucleares Huérfanos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The human monocytic THP-1 cell line differentiates into macrophage-like cells that secrete apo E after addition of PMA. Using this model, we studied the time course of apo E transcriptional activation and secretion in relation with the expression of nuclear receptors. Upon treatment with PMA, apo E mRNA and protein secretion were triggered with the concomitant increase of LXRalpha, PPARgamma, and PPARbeta mRNA expression levels. PPARalpha was downregulated, RXRalpha expression was unchanged, and RARalpha and VDR showed only transient increases. FXR and SXR transcripts were not detectable. Specific agonists were used to investigate the functional role of these nuclear receptors upon apo E secretion. The LXRalpha ligands T0901317 and 22(R)-hydroxycholesterol were the most potent apo E inducers, followed by the PPARgamma agonist BRL49653. The PPARalpha agonist Wy14,643 was inactive and 1alpha,25-dihydroxyvitamin D3, 9-cis-retinoic acid and all-trans-retinoic acid decreased apo E secretion. Thus, during PMA-induced THP-1 differentiation, there is a sequential and coordinate regulation of apo E and nuclear receptor transcription.
Asunto(s)
Apolipoproteínas E/metabolismo , Diferenciación Celular/fisiología , Macrófagos/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Macrófagos/citología , Ésteres del Forbol/farmacología , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: To study the human pharmacokinetics and in vitro cytotoxicity of Apomine, an p.o. administered, nonmyelosuppressive agent that selectively inhibits cell proliferation and induces tumor cell apoptosis through the farnesoid X receptor. EXPERIMENTAL DESIGN: Seven solid cancer patients who participated in an ongoing Phase I study of Apomine and received the starting dose level of 125 mg/m(2)/day x 14 days every 3 weeks underwent a pharmacokinetic study on day 14 of the first course. Plasma concentrations of Apomine were assayed with a Hewlett Packard gas chromatograph using a nitrogen phosphorus detector and HP-5 15m x 0.32-mm column. Fresh human ovarian cancer tumor samples were obtained during initial exploratory laparotomy from 35 chemotherapy-naive, advanced stage epithelial ovarian cancer patients. Tumor samples were tested for sensitivity to Apomine, carboplatin, cisplatin, paclitaxel, and topotecan using an in vitro clonogenic [(3)H]thymidine end point assay. RESULTS: Pharmacokinetic analysis revealed a mean Apomine plasma C(max) of 16.4 +/- 9.1 microg/ml (29.1 microM), a mean plasma AUC(0--12 h) of 173.4 +/- 105 microg. h/ml (308 microM. h), and a mean t(1/2 (24--192 h)) of 156.2 +/- 42.9 h. In vitro assay results showed that 63 and 91% of the ovarian cancers were sensitive (i.e., >70% inhibition of tumor cell growth) to Apomine at concentrations of 10 and 20 microM. The sensitivity rates were 91% for carboplatin (270 microM), 88% for cisplatin (33 microM), 41% for paclitaxel (5.9 microM), and 85% for topotecan (2.2 microM). CONCLUSIONS: These in vitro assay results, taken together with our preliminary plasma pharmacokinetic data, suggest that Apomine should be clinically active at the 125 mg/m(2) dose level.
Asunto(s)
Antineoplásicos/farmacocinética , Difosfonatos/farmacocinética , Neoplasias/metabolismo , Administración Oral , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Difosfonatos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Neoplasias/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
The orphan nuclear receptors FXR and LXRalpha have become challenging targets for the discovery of new therapeutic agents. Bile acids and hydroxysterol intermediates are the respective natural ligands of these two structurally and functionally closely related receptors. Both FXR and LXRalpha; are thought to play a major role in the control of cholesterol catabolism by regulating the expression of cholesterol 7alpha-hydroxylase, the rate limiting enzyme of bile acid synthesis. Reverse cholesterol transport might also be affected by FXR and LXR since they control the expression of PLTP and CETP, two proteins involved in the transfer of phospholipid, cholesterol and cholesteryl esters among plasma lipoproteins. A new class of potent synthetic activators of FXR, the 1,1-bisphosphonate esters, has been discovered which up regulate the Intestinal Bile Acid Binding Protein gene (I-BABP) as demonstrated for chenodeoxycholic acid, however there are no known synthetic activators yet identified for LXRalpha. The evaluation of FXR as a potential target for the development of drugs affecting plasma cholesterol can take advantage of the fact that the activators of FXR (farnesol, bile acids and the 1,1-bisphosphonate esters) have been studied in various in vitro and in vivo models. Administration of chenodeoxycholic acid to animals and man did not result in the increase in plasma cholesterol expected from a decrease in cholesterol 7alpha-hydroxylase expression. Like farnesol, the 1,1-bisphosphonate esters increase the rate of degradation of HMGCoA reductase and have the unexpected property of inducing hypocholesterolemia in normal animals. The natural and synthetic FXR agonists trigger differentiation, inhibit cell proliferation and are potent inducers of apoptosis. The 1,1-bisphosphonate ester SR-45023A (Apomine) is presently being developed as an antineoplastic drug.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Metabolismo de los Lípidos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Anticolesterolemiantes/farmacología , Ácidos y Sales Biliares/metabolismo , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/fisiología , Proteínas de Unión al ADN/fisiología , Diseño de Fármacos , Humanos , Receptores X del Hígado , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/química , Factores de Transcripción/fisiologíaRESUMEN
Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25-hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase-3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol.
Asunto(s)
Anticolesterolemiantes/farmacología , Antineoplásicos/farmacología , Apoptosis , Difosfonatos/farmacología , Farnesol/farmacología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Hidroxicolesteroles/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Ácido Mevalónico/metabolismo , Imitación Molecular , Simvastatina/farmacología , Terpenos/metabolismo , Células Tumorales CultivadasRESUMEN
Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Difosfonatos/farmacología , Receptores Nicotínicos/metabolismo , Animales , Unión Competitiva , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio , Membrana Celular/metabolismo , Diltiazem/farmacología , Cinética , Matemática , Nifedipino/farmacología , Ratas , Receptores Nicotínicos/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis.