RESUMEN
PURPOSE. Topical biphasic vesicle delivery system encapsulating interferon alpha (IFN α) was developed as an alternative to injections used to treat human papillomavirus (HPV) infections. METHODS. Biphasic lipid vesicles encapsulating increasing doses of IFN α (biphasic IFN α) were characterized for encapsulation efficiency, size, zeta potential and vesicle structure by centrifugation, dynamic light scattering, confocal microscopy and small-angle x-ray scattering. Biphasic IFN-α delivery into human skin in vivo and topical efficacy in patients with genital warts were evaluated. RESULTS. Average encapsulation efficiency of IFN α was 81-91%. The average particle size was 1000-1100 nm and zeta potential +70 to +78 mV. After application of 5, 15 and 40MU/g biphasic IFN α formulation in a topical patch on the upper inner arm in healthy volunteers, skin IFN α levels increased to 120±30, 380±60 and 400±80 IU/mg protein in skin homogenates (n=5, 5, and 7), respectively. Topical application of biphasic IFN α (1 MU/dose) twice daily for two weeks in a pilot study with 12 patients with external condylomata acuminata resulted in a decrease in lesion size, in 2',5'-oligoadenylate synthetase activity and in tissue viral load. CONCLUSIONS. Biphasic vesicles delivered clinically significant levels of IFN α across intact human skin and elicited marked therapeutic effect in patients.
Asunto(s)
Condiloma Acuminado/tratamiento farmacológico , Interferón-alfa/administración & dosificación , Interferón-alfa/química , 2',5'-Oligoadenilato Sintetasa/metabolismo , Administración Tópica , Adolescente , Adulto , Condiloma Acuminado/metabolismo , Condiloma Acuminado/virología , Femenino , Papillomavirus Humano 11/aislamiento & purificación , Papillomavirus Humano 6/aislamiento & purificación , Humanos , Interferón-alfa/sangre , Interferón-alfa/farmacocinética , Liposomas/administración & dosificación , Liposomas/química , Liposomas/metabolismo , Masculino , Persona de Mediana Edad , Tamaño de la Partícula , Proyectos Piloto , Piel/efectos de los fármacos , Piel/enzimología , Piel/metabolismo , Carga Viral/efectos de los fármacos , Adulto JovenRESUMEN
Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N-myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI-TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose-dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross-reacted with both anti-NIP71 and anti-HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/química , Proteínas de Neoplasias/química , Homología de Secuencia de Aminoácido , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The cutaneous delivery pathway through the lymphatics of a novel transdermal lipid-based delivery system (biphasic vesicles), which was previously shown to deliver sustained physiological levels of basal insulin in a pain-free manner across the skin, was evaluated in a diabetic rat model. Transdermal patches (one per rat) containing insulin in biphasic vesicles (1-10 mg recombinant human insulin dose) were applied to the shaved abdominal skin of streptozotocin-induced diabetic rats for 73 h. Blood glucose was monitored approximately every 2-10 h using a Lifescan glucose meter. Inguinal lymph node insulin levels were analysed by ELISA. Insulin in the lymph nodes increased in a dose- and time-dependent manner. Maximal transdermal insulin concentrations in the lymph nodes were observed with both 140 IU (5 mg: 43.0 +/- 18.0 microIU mg(-1) (mean +/- s.e.m., n = 4)) and 280 IU (10 mg: 48.0 +/- 19.6 microIU mg(-1) (mean +/- s.e.m., n = 4)) doses of recombinant insulin at t = 73 h. The level of insulin in the lymph nodes after subcutaneous injection of 1 mg insulin at the peak blood glucose response was 35.8 microIU mg(-1) (n = 2), before falling to 0.35 microIU mg(-1) by t = 48 h (n = 2). The lymphatics is involved in the transdermal insulin delivery by biphasic vesicles. This is the first report on the lymphatic transport of a protein after non-invasive topical application on the skin.
Asunto(s)
Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Insulina/administración & dosificación , Insulina/farmacocinética , Administración Cutánea , Administración Tópica , Animales , Glucemia/análisis , Diabetes Mellitus/tratamiento farmacológico , Modelos Animales de Enfermedad , Inyecciones Subcutáneas , Sistema Linfático/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Noninvasive transdermal insulin delivery could provide diabetic patients with sustained physiological levels of basal insulin in a pain-free manner. We have developed a novel transdermal lipid-based system (Biphasix) suitable for macromolecule delivery across the skin. The objective of this study was to evaluate the pharmacological effects of the Biphasix-insulin delivery system in a diabetic rat model. Transdermal patches (one per animal) containing Biphasix-insulin formulation (10 mg of recombinant human insulin dose) were applied to the shaved abdominal skin of streptozotocin-induced diabetic rats for 48 h. Blood glucose was monitored every 2-4 h using a Lifescan glucose meter. Serum insulin levels were analysed by enzyme-linked immunosorbent assay. A decrease in blood glucose of 43.7 +/- 3.8% (mean +/- SEM, n = 25) was observed compared with initial blood glucose levels. The duration of the response was 51.5 +/- 3.7 h (mean +/- SEM, n = 25). Serum insulin after application of the transdermal Biphasix-insulin patch was 20.08 +/- 5.44 micro IU/mL (mean +/- SEM, n = 13) during the steady state, which was not statistically different from the insulin levels obtained 2 h after subcutaneous injection of 1 mg of recombinant human insulin solution. Insulin bioavailability from the transdermal Biphasix-insulin patches was 21.5 +/- 6.9% (mean +/- SEM, n = 13) based on serum insulin and 39.5 +/- 8.5% (mean +/- SEM, n = 25) based on the pharmacodynamic blood glucose-lowering effects. The Biphasix system successfully delivered insulin transdermally, as evidenced by a significant sustained decrease in blood glucose in diabetic rats, with a corresponding increase in serum insulin. These results support the feasibility of developing a transdermal insulin patch for human applications.