RESUMEN
For efficient intranasal transport of parathyroid hormone (1-34) [PTH(1-34)], there is a great medical need to investigate permeation enhancers for intranasal formulations. In this study, the development of PTH(1-34) intranasal formulations was conducted. Based on conformation and chemical stability studies, the most preferable aqueous environment was determined to be 0.008 M acetate buffer solution (ABS). Subsequently, citric acid and Kolliphor® HS·15 were compared as permeation enhancers. The mechanisms of action of citric acid and Kolliphor® HS·15 were investigated using an in vitro model of nasal mucosa, and Kolliphor® HS·15 led to higher permeability of fluorescein isothiocyanate-labeled PTH(1-34) (FITC-PTH) by enhancing both the transcellular and paracellular routes. Moreover, citric acid showed severe mucosal toxicity resulting in cilia shedding, while Kolliphor® HS·15 did not cause obvious mucosa damage. Finally, Kolliphor® HS·15 was studied as a permeation enhancer using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The results showed that 5% and 10% Kolliphor® HS·15 increased the bioavailability of PTH(1-34) to 14.76% and 30.87%, respectively. In conclusion, an effective and biosafe PTH(1-34) intranasal formulation was developed by using 10% Kolliphor® HS·15 as a permeation enhancer. Intranasal formulations with higher concentrations of Kolliphor® HS·15 for higher bioavailability of PTH(1-34) could be further researched.
Asunto(s)
Excipientes/química , Mucosa Nasal/metabolismo , Hormona Paratiroidea/administración & dosificación , Administración Intranasal , Animales , Anuros , Disponibilidad Biológica , Cromatografía Liquida , Ácido Cítrico/química , Ácido Cítrico/toxicidad , Excipientes/toxicidad , Femenino , Masculino , Hormona Paratiroidea/farmacocinética , Hormona Paratiroidea/toxicidad , Permeabilidad , Polietilenglicoles/química , Polietilenglicoles/toxicidad , Ratas , Ratas Sprague-Dawley , Estearatos/química , Estearatos/toxicidad , Espectrometría de Masas en TándemRESUMEN
Prolonged treatment with human parathyroid hormone (hPTH) in rats results in development of bone tumors, though this finding has not been supported by clinical experience. The PTH type 1 receptor agonist abaloparatide, selected for its bone anabolic activity, is under clinical development to treat postmenopausal women with osteoporosis. To determine the carcinogenic potential of abaloparatide, Fischer (F344) rats were administered SC daily abaloparatide at doses of 0, 10, 25, and 50 µg/kg or 30 µg/kg hPTH(1-34) as a positive control for up to 2 years. Robust increases in bone density were achieved at all abaloparatide doses and with hPTH(1-34). Comprehensive histopathological analysis reflected a comparable continuum of proliferative changes in bone, mostly osteosarcoma, in both abaloparatide and hPTH(1-34) treated rats. Comparing the effects of abaloparatide and hPTH(1-34) at the 25 and 30 µg/kg respective doses, representing similar exposure multiples to the human therapeutic doses, revealed similar osteosarcoma-associated mortality, tumor incidence, age at first occurrence, and metastatic potential. There were no increases in the incidence of non-bone tumors with abaloparatide compared to vehicle. Thus, near life-long treatment with abaloparatide in rats resulted in dose and time dependent formation of osteosarcomas, with a comparable response to hPTH(1-34) at similar exposure.
Asunto(s)
Conservadores de la Densidad Ósea/toxicidad , Neoplasias Óseas/inducido químicamente , Osteosarcoma/inducido químicamente , Proteína Relacionada con la Hormona Paratiroidea/toxicidad , Hormona Paratiroidea/toxicidad , Animales , Conservadores de la Densidad Ósea/administración & dosificación , Neoplasias Óseas/epidemiología , Femenino , Humanos , Incidencia , Osteosarcoma/epidemiología , Hormona Paratiroidea/administración & dosificación , Proteína Relacionada con la Hormona Paratiroidea/administración & dosificación , Ratas , Ratas Endogámicas F344RESUMEN
Toxicological studies have demonstrated that intermittent PTH1-34 treatment is associated with an increased incidence of osteosarcoma in Fischer 344 rats. Comet and micronucleus (MN) tests, standard methods to evaluate genotoxic potential of drugs, were used to detect DNA and chromosome breaks, respectively, after PTH1-34 treatment. MC3T3 cells, primary osteoblast calvarial cells, and human osteoblasts were treated with PTH1-34 (50 and 100 nM) for 6 h/day for 21 days to mimic intermittent administration. Genotoxic assays were performed at 6 h and 7, 14, and 21 days. Osteoblasts extracted from bone marrow of mice treated with daily subcutaneous PTH1-34 injections (20 and 40 µg/kg) for 10 weeks as well as Hep-2, HeLa, and Hep-G2 cells were also tested. We observed a significant increase in DNA lesions and MN prevalence in human and murine osteoblasts treated with PTH1-34 compared to controls (P < 0.01). The effect observed in vitro and confirmed in vivo was time- and dose-dependent. For nonosteoblastic Hep-2 and HeLa cells we observed increased DNA damage and MN prevalence only later in the course of the protocol, after 21 days of treatment (P < 0.01). In Hep-G2 cells intermittent PTH1-34 did not induce DNA damage or chromosome breaks. Our results demonstrated that intermittent PTH increases DNA and chromosome breaks in osteoblasts. This genotoxic effect is attenuated in nonosteoblastic cells, and the ability to induce DNA damage is lost in cells with detoxification properties (HepG2 cells) tested in vitro.
Asunto(s)
Carcinógenos/toxicidad , Rotura Cromosómica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/toxicidad , Animales , Animales Recién Nacidos , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Daño del ADN/genética , Células HeLa , Células Hep G2 , Humanos , Ratones , Células 3T3 NIH , Osteoblastos/metabolismoAsunto(s)
Pruebas de Carcinogenicidad/métodos , Neoplasias Experimentales/inducido químicamente , Hormona Paratiroidea/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Incidencia , Masculino , Neoplasias Experimentales/epidemiología , Hormona Paratiroidea/farmacocinética , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidad , Medición de Riesgo , Teriparatido/toxicidadRESUMEN
The carcinogenic potential of human parathyroid hormone 1-84 (PTH) was assessed by daily subcutaneous injection (0, 10, 50, 150 microg/kg/day) for 2 years in Fischer 344 rats. Histopathological analyses were conducted on the standard set of soft tissues, tissues with macroscopic abnormalities, selected bones, and bones with abnormalities identified radiographically. All PTH doses caused widespread osteosclerosis and significant, dose-dependent increases in femoral and vertebral bone mineral content and density. In the mid-and high-dose groups, proliferative changes in bone increased with dose. Osteosarcoma was the most common change, followed by focal osteoblast hyperplasia, osteoblastoma, osteoma and skeletal fibrosarcoma. The incidence of bone neoplasms was comparable in control and low-dose groups providing a noncarcinogenic dose for PTH of 10 microg/kg/day at a systemic exposure to PTH that is 4.6-fold higher than for a 100 microg dose in humans. The ability of PTH to interact with and balance the effects of both the PTH-1 receptor and the putative C-terminal PTH receptor, may lead to the lower carcinogenic potential observed with PTH than reported previously for teriparatide.
Asunto(s)
Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/toxicidad , Animales , Área Bajo la Curva , Densidad Ósea/efectos de los fármacos , Neoplasias Óseas/inducido químicamente , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/patología , Densitometría , Femenino , Fibrosarcoma/inducido químicamente , Fibrosarcoma/diagnóstico por imagen , Fibrosarcoma/patología , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/diagnóstico por imagen , Hiperplasia/patología , Inyecciones Subcutáneas , Masculino , Osteoblastoma/inducido químicamente , Osteoblastoma/diagnóstico por imagen , Osteoblastoma/patología , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteosarcoma/inducido químicamente , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/patología , Osteosclerosis/inducido químicamente , Osteosclerosis/diagnóstico por imagen , Osteosclerosis/patología , Hormona Paratiroidea/farmacocinética , Radiografía , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidad , Factores SexualesAsunto(s)
Conservadores de la Densidad Ósea/efectos adversos , Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/patología , Calcio/toxicidad , Difosfonatos/efectos adversos , Enfermedades Maxilomandibulares/inducido químicamente , Osteonecrosis/inducido químicamente , Hormona Paratiroidea/toxicidad , Adulto , Anciano , Anciano de 80 o más Años , Conservadores de la Densidad Ósea/uso terapéutico , Neoplasias Óseas/secundario , Calcio/sangre , Difosfonatos/uso terapéutico , Femenino , Humanos , Persona de Mediana Edad , Hormona Paratiroidea/sangreRESUMEN
UNLABELLED: We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. INTRODUCTION: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. MATERIALS AND METHODS: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 microg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. microCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. RESULTS AND CONCLUSIONS: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.
Asunto(s)
Remodelación Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Animales , Fenómenos Biomecánicos , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Femenino , Ovariectomía , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/toxicidad , Porosidad , Radiografía , Ratas , Ratas Sprague-DawleyRESUMEN
The purpose of this study was to evaluate whether the natriuresis and polyuria seen in parathyroid hormone (PTH)-induced hypercalcemia are associated with dysregulation of renal Na transporters. Rats were infused with three different doses of human PTH [PTH (1-34); 7.5, 10, and 15 microg.kg(-1).day(-1) s.c.] or vehicle for 48 h using osmotic minipumps. The rats treated with PTH developed significant hypercalcemia (plasma total calcium levels: 2.71 +/- 0.03, 2.77 +/- 0.02, and 3.42 +/- 0.06 mmol/l, respectively, P < 0.05 compared with corresponding controls). The rats with severe hypercalcemia induced by high-dose PTH developed a decreased glomerular filtration rate (GFR), increased urine output, reduced urinary osmolality, increased urinary Na excretion, and fractional excretion of Na. This was associated with downregulation (calculated as a fraction of control levels) of whole kidney expression of type 2 Na-P(i) cotransporter (NaPi-2; 16 +/- 6%), type 3 Na/H exchanger (NHE3; 42 +/- 7%), Na-K-ATPase (55 +/- 2%), and bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1; 25 +/- 4%). In contrast, an upregulation of the Ca(2+)-sensing receptor (CaR) was observed. Rats treated with moderate-dose PTH exhibited unchanged GFR but decreased urinary concentration. The whole kidney expression of NHE3 (52 +/- 8%) and NaPi-2 (26 +/- 5%) was persistently decreased, whereas BSC-1 and Na-K-ATPase protein levels were not altered. CaR expression was also increased. Moreover, rats treated with low-dose PTH showed very mild hypercalcemia but unchanged GFR, normal urinary concentration, and unchanged expression of Na transporters and CaR. In conclusion, the reduced expression of major renal Na transporters is likely to play a role in the increased urinary Na excretion and decreased urinary concentration in rats with PTH-induced hypercalcemia. Moreover, the increase in the CaR in the thick ascending limb (TAL) may indicate a potential role of the CaR in inhibiting Na transport in the TAL.
Asunto(s)
Hipercalcemia/metabolismo , Bombas Iónicas/metabolismo , Riñón/metabolismo , Hormona Paratiroidea/toxicidad , Sodio/metabolismo , Animales , Creatinina/orina , Relación Dosis-Respuesta a Droga , Tasa de Filtración Glomerular , Hipercalcemia/inducido químicamente , Hipercalcemia/diagnóstico , Masculino , Natriuresis , Poliuria/inducido químicamente , Ratas , Ratas Wistar , Receptores Sensibles al Calcio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II , Simportadores de Cloruro de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Simportadores/metabolismoRESUMEN
Anti-bone resorption properties of the Korean herbal formulation, Honghwain (HHI; Carthamus tinctorius L. seed) was biochemically investigated. On processing bone metabolism, PGE2 accelerated production of IL-1beta in fetal mouse osteoblast and stimulated physiological activation substance, IL-1beta. The novel class of Src tyrosine kinase inhibitors, Herbimycin A (HERB) and HHI reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta, TNF-alpha and IL-6. HHI inhibited in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. HHI dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase (s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, HHI as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Carthamus/química , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/farmacología , Isoenzimas/metabolismo , Extractos Vegetales/farmacología , Antagonistas de Prostaglandina/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Benzoquinonas , Resorción Ósea/inducido químicamente , Resorción Ósea/patología , Huesos/patología , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Citocinas/farmacología , Femenino , Hipercalcemia/sangre , Hipercalcemia/tratamiento farmacológico , Isoenzimas/biosíntesis , Lactamas Macrocíclicas , Masculino , Proteínas de la Membrana , Ratones , Osteoblastos/efectos de los fármacos , Osteoporosis/inducido químicamente , Osteoporosis/prevención & control , Ovariectomía , Hormona Paratiroidea/antagonistas & inhibidores , Hormona Paratiroidea/toxicidad , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Quinonas/farmacología , ARN Mensajero/biosíntesis , Rifabutina/análogos & derivados , Semillas/químicaRESUMEN
The anemia associated with renal failure is largely due to inappropriate erythropoietin production. There is also good evidence, however, that substances present in uremic serum can inhibit erythropoiesis, although the exact identity of these substances and the mechanism(s) by which they exert this effect remain obscure. Candidates that have been suggested to play a role in uremic inhibition of erythropoiesis include the polyamines (such as spermine, spermidine, putrescine, and cadaverine), parathyroid hormone, and some of the inflammatory cytokines. The potential role of each of these inhibitory substances is discussed in this article.
Asunto(s)
Anemia/etiología , Fallo Renal Crónico/complicaciones , Toxinas Biológicas/sangre , Uremia/sangre , Uremia/complicaciones , Animales , Citocinas/sangre , Citocinas/toxicidad , Eritropoyesis/efectos de los fármacos , Humanos , Técnicas In Vitro , Hormona Paratiroidea/sangre , Hormona Paratiroidea/toxicidad , Poliaminas/sangre , Poliaminas/toxicidad , Toxinas Biológicas/toxicidadRESUMEN
Adverse effects of an active fragment of parathyroid hormone (PTH(1 - 34)), a blood Ca(2+) level-regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 microM PTH(1 - 34) for 60 min resulted in a gradual increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH(1 - 34) at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose-dependent over the range of 0.1 pM to 0.1 microM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH(39 - 84), or by an active fragment of PTH-related peptide (PTHrP(1 - 34)), an intrinsic ligand of the brain PTH receptor. The PTH(1 - 34)-induced adverse effects were significantly inhibited by co-administration of 10 microM nifedipine, an L-type Ca(2+) channel blocker, but not by co-administration of blockers of the other types of Ca(2+) channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca(2+) overloading via activation of dihydropyridine-sensitive Ca(2+) channels, and suggests that PTH may be a risk factor for senile dementia. British Journal of Pharmacology (2000) 129, 21 - 28
Asunto(s)
Hipocampo/efectos de los fármacos , Hormona Paratiroidea/toxicidad , Fragmentos de Péptidos/toxicidad , Proteínas , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Femenino , Colorantes Fluorescentes , Fura-2 , Procesamiento de Imagen Asistido por Computador , Masculino , Nifedipino/farmacología , Técnicas de Cultivo de Órganos , Hormona Paratiroidea/biosíntesis , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/farmacología , Propidio/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptores de Hormona Paratiroidea/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We previously found that a methanolic extract of the stems of Sambucus sieboldiana inhibited bone resorption in organ culture. In this study, we further fractionated the methanol extract guided by the activity towards bone resorption stimulated by parathyroid hormone (PTH) in vitro. The ethyl acetate fraction (EtOAc Fr.) of the methanolic extract inhibited PTH-stimulated bone resorption of neonatal mouse bones, and the inhibitory activity was more potent than those of other fractions. Oral administration of the EtOAc Fr. (50 and 100 mg/kg/d) to ovariectomized (OVX) rat prevented the decrease in bone mineral density (BMD) of the lumbar (L2-4) vertebra, indicating that the EtOAc Fr. is effective in vivo. Furthermore, the EtOAc Fr. (50, 100 and 150 mg/kg/d) decreased the serum calcium level elevated in low calcium dietary rats. The phenolic constituents of the EtOAc fraction were examined for their inhibitory effect on bone resorption stimulated by PTH in neonatal mouse bone. Among them, vanillic acid, vanillin and coniferyl alcohol showed significant inhibitory effects on bone resorption. Of the compounds examined, vanillic acid was found to have a significant inhibitory effect on the decrease of BMD in OVX mice. Therefore, the EtOAc Fr. of S. sieboldiana showed a suppressive effect on bone resorption in vitro and in vivo. In addition, the inhibitory effects of the EtOAc Fr. on bone resorption may be at least partly due to the inhibitory action of vanillic acid.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Plantas Medicinales/química , Acetatos , Animales , Benzaldehídos/farmacología , Densidad Ósea/efectos de los fármacos , Resorción Ósea/inducido químicamente , Calcio/sangre , Calcio/deficiencia , Calcio de la Dieta/administración & dosificación , Ratones , Ovariectomía , Hormona Paratiroidea/toxicidad , Fenoles/farmacología , Extractos Vegetales/farmacología , Ratas , Ratas Wistar , Solventes , Factores de Tiempo , Ácido Vanílico/farmacologíaRESUMEN
We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na(+)-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production, Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gs alpha coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , AMP Cíclico/biosíntesis , Diterpenos , Riñón/efectos de los fármacos , Hormona Paratiroidea/toxicidad , Fosfatos/metabolismo , Proteína Quinasa C/metabolismo , Terpenos/toxicidad , Animales , Sitios de Unión , Transporte Biológico Activo , División Celular/efectos de los fármacos , Células Cultivadas , Toxina del Cólera/toxicidad , Colforsina/toxicidad , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Riñón/citología , Riñón/metabolismo , Zarigüeyas , Isótopos de Fósforo , Sodio/farmacologíaRESUMEN
BIM-44002, a pure competitive antagonist of parathyroid hormone (PTH), has a high affinity for the PTH/PTHrP receptor in vitro, and can completely inhibit the actions of a PTH agonist in rats in vivo. Toxicology studies in rats and dogs showed BIM-44002 to be devoid of any adverse effects. Therefore we undertook an investigation to evaluate the potential utility of BIM-44002 in lowering elevated serum calcium in three patients with primary hyperparathyroidism. BIM-44002 was administered by continuous intravenous infusion at dosages of 100 microg/hour (370 nmol/hour) for 12 hours, followed by 200 microg/hour for 12 hours, followed by 400 microg/hour for 12 hours. Vital signs and serum ionized and total calcium were monitored hourly and for 3 hours after cessation of the infusion. Blood for PTH determinations was obtained at the same time points. Serum calcium and PTH did not change during and after the infusion of the antagonist. No subject experienced any adverse reactions to the infusion of the antagonist. We conclude that although the PTH antagonist BIM-44002 was effective both in vitro and in vivo in animals, and it was safe in humans, it was not able to lower serum calcium in patients with hyperparathyroidism. Possible reasons for lack of clinical efficacy are discussed.
Asunto(s)
Calcio/sangre , Hipercalcemia/sangre , Hiperparatiroidismo/sangre , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Perros , Femenino , Humanos , Infusiones Intravenosas , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/toxicidad , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Paratiroidea/antagonistas & inhibidoresRESUMEN
Exposure to cadmium (Cd) causes skeletal impairments, such as osteoporosis and osteomalacia, in many mammalian species, including humans. There is, however, some controversy about the mechanism of action of these Cd-induced skeletal effects, although both a direct influence on bone cells and effects that are secondary to renal damage caused by the metal have been demonstrated. In the present study, we cultured calvarial bones from neonatal mice and exposed them to Cd to study the effects of the metal on calcium release and on the activity of some enzymes of importance for bone resorption and bone formation. Cd dose-dependently stimulated calcium release from the bones. Maximal release was noted at Cd concentrations of 0.4-0.8 microM, which was similar to the level of release in the presence of maximal stimulatory concentrations of parathyroid hormone (10 nM) and prostaglandin E2 (10 microM). Cykloheximide (1 microM) inhibited calcium release elicited by Cd, prostaglandin E2 and parathyroid hormone. Cd-induced calcium release was linearly increased from 24 to 72 hr of culture. Production of prostaglandin E2 by the bone specimens was dose-dependently stimulated by Cd and inhibited by 1 microM indomethacin. Cd-induced calcium release was inhibited by acetazolamide (100 microM), indomethacin (1 microM) and ibuprofen (10 microM). Prostaglandin E2-stimulated calcium release was not inhibited by indomethacin. Exposure to 32 microM Cd, present during a 48-hr incubation period, significantly decreased prostaglandin E2-stimulated calcium release from 38.9% to 29.8%. Calcium release induced by parathyroid hormone was more sensitive to inhibition by the metal (i.e., Cd concentrations of 0.2 and 32 microM decreased the release from 37.7% to 31% and 19%, respectively). Cd present in the culture medium during a 48-hr incubation dose-dependently inhibited the activity of alkaline phosphatase and tartrate-resistant acid phosphatase in the bones but did not influence the activity of carbonic anhydrase. We conclude that Cd has a direct stimulatory effect on bone resorption, and this effect is dependent on prostaglandin production and also on protein synthesis. On the other hand, Cd also has an inhibitory effect on bone resorption (i.e., resorption is inhibited by higher concentrations of the metal). Moreover, Cd may impair bone formation by impeding the activity of alkaline phosphatase.
Asunto(s)
Cadmio/toxicidad , Calcio/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Cráneo/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Biomarcadores/análisis , Resorción Ósea , Anhidrasas Carbónicas/metabolismo , Cicloheximida/farmacología , Dinoprostona/toxicidad , Relación Dosis-Respuesta a Droga , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Hormona Paratiroidea/toxicidad , Cráneo/enzimología , Fosfatasa Ácida TartratorresistenteRESUMEN
In vitro studies of parathyroid hormone (PTH) structure and function have suggested that the anabolic effect of PTH on bone requires the presence of amino acid residues 28-34 (domains for protein kinase C activation and mitogenic activity), but not amino acid residues 1-7 (adenylate cyclase activation domain). We have tested this hypothesis with in vivo studies of human PTH (hPTH) analogs. Serum biomarkers and selected histomorphometric parameters of bone formation and resorption were assessed in adult, female, Sprague-Dawley rats following 19 daily injections of vehicle, 10 micrograms/kg body weight (bw) of hPTH(1-38), or a dose range of 10, 40, and 100 micrograms/100 g bw of hPTH(2-38) or hPTH(3-38). Treatment with hPTH(1-38) increased serum osteocalcin, the percentage of osteoblast surface, percentage of osteoid surface, percentage of bone volume, trabecular thickness, and bone formation rate, while it decreased the percentage of osteoclast surface. The hPTH(2-38) fragment exhibited 10%-25% of the in vivo anabolic activity of hPTH(1-38), while it had no effect on the percentage of osteoclast surface. The hPTH(3-38) fragment exhibited no biological activity on bone. In contrast, serum INS-PTH (intact-N-terminal specific PTH) levels were similarly and significantly increased above control in rats treated with hPTH(1-38), hPTH(2-38), or hPTH(3-38) at the same dose. This preliminary finding suggests that the differential activity of these peptides on bone is not due to differences in the circulating level of immunoreactive PTH (intact and amino-terminal fragments of PTH from endogenous and exogenous sources) several hours after PTH injection. However, we can draw no conclusion regarding the relative clearance rates of these peptides. Last, because hPTH(3-38) was without any detectable biological activity on rat bone in vivo, its mitogenic activity was confirmed in two osteoblast-like cell lines. In summary, the anabolic effect of hPTH(1-38) on bone in vivo was (1) diminished by removal of amino acid residue 1, and (2) abolished by the removal of amino acid residues 1 and 2. Although these findings suggest that the therapeutic benefits of exogenous PTH administration may depend upon activation of not only protein kinase C, but also adenylate cyclase, they do not rule out a differential PTH response due to other causes, e.g., metabolic inactivation.
Asunto(s)
Biomarcadores/sangre , Densidad Ósea/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Hormona Paratiroidea/farmacología , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Resorción Ósea/prevención & control , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Mitógenos/toxicidad , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteocalcina/sangre , Hormona Paratiroidea/sangre , Hormona Paratiroidea/química , Hormona Paratiroidea/toxicidad , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Although colony stimulating factor-1 (CSF-1) plays a key role in osteoclast recruitment, studies examining the effect of CSF-1 on mature osteoclasts indicate that it may directly inhibit bone resorption by isolated rat osteoclasts. To define further CSF-1's role in bone remodeling, we examined the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts, respectively. We first assessed whether PTH increases CSF-1 production from bone in organ culture by examining conditioned medium from 19-day-old fetal rat long bones in a mitogenesis assay employing a CSF-1-responsive cell line, CRX-1. Conditioned medium from untreated bones induced a titratable increase in CRX-1 cell proliferation, and treatment of bones with PTH for 72 h caused a significant increase in mitogenic activity. CSF-1 antiserum caused a significant decrease in mitogenic activity in conditioned medium, indicating that bone in organ culture produces CSF-1 constitutively and in response to PTH. To examine bone-derived CSF-1's role in bone resorption, we examined the effect of neutralizing antisera to CSF-1 on basal and PTH-induced bone resorption in the fetal rat long bone assay, which reflects activation of mature osteoclasts. Anti-CSF-1 caused a significant increase in unstimulated and PTH-induced bone resorption compared with control. By contrast, in the fetal mouse metacarpal assay, which examines proliferation and recruitment of osteoclast progenitors and precursors, anti-CSF-1 caused significant inhibition of PTH related protein (PTHrP)-induced bone resorption after 3 and 6 days of incubation. Consistent with these findings, histological examination of cultured 17-day-old fetal metacarpals demonstrated that anti-CSF-1 inhibits the formation of tartrate-resistant acid phosphatase-positive osteoclasts in PTHrP-treated explants, whereas it has no effect on unstimulated bones. We conclude that bone-derived CSF-1 may have a dual role in PTH/PTHrP-induced bone resorption by enhancing the appearance of osteoclast precursors while restraining the resorptive function of mature osteoclasts.
Asunto(s)
Resorción Ósea/inducido químicamente , Factor Estimulante de Colonias de Macrófagos/metabolismo , Osteoclastos/citología , Hormona Paratiroidea/toxicidad , Teriparatido/toxicidad , Fosfatasa Ácida/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Células de la Médula Ósea , Resorción Ósea/metabolismo , Femenino , Isoenzimas/metabolismo , Metacarpo/citología , Ratones , Técnicas de Cultivo de Órganos , Osteoclastos/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Células Madre/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.