RESUMEN
The role of plasma membrane transporters in the nephrotoxicity of two antiretroviral drugs, cidofovir and tenofovir, was studied in primary cultures of human proximal tubular (hPT) cells. Cells were grown on Transwell filter inserts to maintain epithelial polarity and access to either the apical or basolateral plasma membrane. The function of relevant membrane transporters, organic anion transporter 1 and 3 (OAT1/3), P-glycoprotein (multidrug resistance protein-1; P-gp or MDR1), and organic cation transporter 2 (OCT2), was validated by measurements of apical-to-basolateral and basolateral-to-apical fluxes of furosemide, digoxin, and metformin, respectively. Acute cytotoxicity of cidofovir (0, 10, 50, 150, or 300⯵M) in the absence or presence of 500⯵M probenecid, tenofovir disoproxil fumarate (0, 20, 90, 180, or 360⯵M) in the absence or presence of 500⯵M probenecid, or cisplatin (0, 20, 90, 180, or 360⯵M) as a positive control in the absence or presence of 500⯵M cimetidine, was assessed after 4-h incubations by determinations of release of lactate dehydrogenase (LDH), γ-glutamyltransferase (GGT), N-acetyl-ß-d-glucosaminidase (NAG), or Kidney Injury Molecule-1 (KIM-1). Cell death generally agreed with each of the four biomarkers, was generally greater when cidofovir or tenofovir was added to the upper compartment, and was markedly diminished in the presence of the appropriate transport inhibitor. Additionally, the extent of cytotoxicity caused by the two antiviral drugs was similar to that caused by cisplatin. The results demonstrate the importance of plasma membrane transport of antiviral drugs to elicit cytotoxicity in the hPT cell.
Asunto(s)
Antivirales/metabolismo , Antivirales/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Transportador 2 de Cátion Orgánico/metabolismo , Anciano , Células Cultivadas , Citotoxinas/metabolismo , Citotoxinas/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.
Asunto(s)
Gota/tratamiento farmacológico , Urato Oxidasa/uso terapéutico , Animales , Rastreo Diferencial de Calorimetría , Perros , Escherichia coli/metabolismo , Semivida , Humanos , Concentración de Iones de Hidrógeno , Cinética , Papio , Polietilenglicoles/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Especificidad por Sustrato , Porcinos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Urato Oxidasa/efectos adversos , Urato Oxidasa/inmunologíaAsunto(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.