RESUMEN
Selective deuterium substitution as a means of ameliorating clinically relevant pharmacokinetic drug interactions is demonstrated in this study. Carbon-deuterium bonds are more stable than corresponding carbon-hydrogen bonds. Using a precision deuteration platform, the two hydrogen atoms at the methylenedioxy carbon of paroxetine were substituted with deuterium. The new chemical entity, CTP-347 [(3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-yloxy)methyl)-4-(4-fluorophenyl)piperidine], demonstrated similar selectivity for the serotonin receptor, as well as similar neurotransmitter uptake inhibition in an in vitro rat synaptosome model, as unmodified paroxetine. However, human liver microsomes cleared CTP-347 faster than paroxetine as a result of decreased inactivation of CYP2D6. In phase 1 studies, CTP-347 was metabolized more rapidly in humans and exhibited a lower pharmacokinetic accumulation index than paroxetine. These alterations in the metabolism profile resulted in significantly reduced drug-drug interactions between CTP-347 and two other CYP2D6-metabolized drugs: tamoxifen (in vitro) and dextromethorphan (in humans). Our results show that precision deuteration can improve the metabolism profiles of existing pharmacotherapies without affecting their intrinsic pharmacologies.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2D6/farmacología , Citocromo P-450 CYP2D6/metabolismo , Paroxetina/farmacología , Animales , Encéfalo/metabolismo , Inhibidores del Citocromo P-450 CYP2D6/farmacocinética , Deuterio , Método Doble Ciego , Interacciones Farmacológicas , Femenino , Humanos , Marcaje Isotópico , Microsomas Hepáticos/metabolismo , Paroxetina/farmacocinética , Paroxetina/uso terapéutico , Ensayo de Unión Radioligante , Ratas , Receptores de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinética , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Sinaptosomas/metabolismo , Tamoxifeno/metabolismoRESUMEN
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 SexualesRESUMEN
Immunoassays utilizing commercial kits designed for diagnostic use can be adapted and validated to meet Good Laboratory Practice (GLP) requirements to support pharmacokinetic (PK) studies. We illustrate in this paper a systematic approach for commercial kit evaluation and GLP-compliant method validation to establish selectivity, sensitivity, linearity, accuracy, precision and stability. Immunoassay kits for human parathyroid hormone (hPTH) quantification from three different vendors were assessed in a side-by-side comparison for their suitability for the PK analysis of recombinant humanPTH (rhPTH) in EDTA plasma. Two immunoradiometric (IRMA) assay kits and one immunoluminometric assay (ILMA) kit were evaluated. Since PTH is present as an endogenous component of human plasma, QC preparation in the biological matrix was handled differently than for a xenobiotic drug compound. The endogenous concentration of PTH was determined in plasma samples from 32 individual lots using the three kits. The lots with the lowest endogenous concentrations of PTH were selected, pooled to form the low QC and spiked with rhPTH to prepare the mid and high QCs. Four evaluation batches were run with each of the three commercial kits to evaluate reference standard linearity, and QC accuracy and precision. Selectivity against PTH peptide fragments PTH(7-84) and PTH(3-84) were assessed by cross-reactivity and accurate spike-recovery to the QC samples at two concentrations. One of the kits was chosen for full method validation because it had the lowest cross-reactivity against hPTH fragments (3-84) and (7-84), a wider dynamic range and the least total error. The accuracy and precision from six validation batches of the QCs were Asunto(s)
Ensayo Inmunorradiométrico/métodos
, Hormona Paratiroidea/sangre
, Juego de Reactivos para Diagnóstico/normas
, Proteínas Recombinantes/sangre
, Estabilidad de Medicamentos
, Almacenaje de Medicamentos
, Humanos
, Ensayo Inmunorradiométrico/instrumentación
, Control de Calidad
, Estándares de Referencia
, Reproducibilidad de los Resultados
, Sensibilidad y Especificidad
RESUMEN
For decades, researchers have tried to identify the primary structures of circulating carboxyl-terminal parathyroid hormone (C-PTH) peptide fragments that may be present at only picomolar levels in human plasma. Although immunoassays and radiosequencing techniques have provided valuable fragment characterizations, no analysis has successfully determined their exact primary structures. In this work, for the first time, four human C-PTH peptide fragments, hPTH(34-84), hPTH(37-84), hPTH(38-84), and hPTH(45-84), have been identified from human plasma using MS-based methods. C-PTH peptide fragments were isolated from plasma samples by immunoaffinity extraction. The eluate was analyzed by capillary LC fractionation followed by MALDI-TOF-MS or by on-line coupling of nano-LC with ESI-TOF-MS. Both the MALDI- and the ESI-based approaches were capable of detecting C-PTH peptide fragments in human plasma at <10 pmol/L. The MALDI-TOF approach was effective in preliminary searches for C-PTH peptide fragments, but the use of high laser power limited the resolution necessary for accurate C-PTH peptide identification. The high mass resolution (10,000) and accuracy (10 ppm) attained by the ESI-TOF approach enabled unambiguous identification of these peptides. The four C-PTH peptide fragments identified in plasma samples from patients with chronic renal insufficiency were also found in the plasma of healthy women receiving recombinant human PTH either by subcutaneous injection or by intravenous infusion. This newly developed analytical capability should greatly enhance the understanding of PTH metabolism and parathyroid gland function.