RESUMEN
Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.
Asunto(s)
Catepsina B/química , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Inmunoconjugados/química , Inmunoglobulina G/análisis , Preparaciones Farmacéuticas/análisis , Antineoplásicos/química , Tampones (Química) , Humanos , Lisina/química , Espectrometría de Masas , Preparaciones Farmacéuticas/química , Polisorbatos/química , Estándares de Referencia , Reproducibilidad de los Resultados , Rayos UltravioletaRESUMEN
Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry-eye disease that affect the vision of millions worldwide. To overcome this challenge, this manuscript explores a novel strategy to reach the ocular surface via receptor-mediated transcytosis across the lacrimal gland (LG), which produces the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR); furthermore, we recently reported a peptide-based nanoparticle (KSI) that targets CAR on liver cells. This manuscript reports the unexpected finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in lumen formed by LG acinar cells. LG transduction with a myosin Vb tail, which is dominant negative towards transcytosis, inhibits lumenal accumulation. Transcytosis of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following administration of KSI nanoparticles. These findings suggest that it is possible to target nanomaterials to the tears by targeting certain receptors on the LG. This design strategy represents a new opportunity to overcome barriers to ocular delivery.
Asunto(s)
Aparato Lagrimal/efectos de los fármacos , Aparato Lagrimal/metabolismo , Nanopartículas/administración & dosificación , Lágrimas/efectos de los fármacos , Transcitosis/efectos de los fármacos , Células Acinares/efectos de los fármacos , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/efectos de los fármacos , Síndromes de Ojo Seco/tratamiento farmacológico , Endocitosis , Femenino , Hepatocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Péptidos/administración & dosificación , Péptidos/química , Polímeros , Cultivo Primario de Células , ConejosRESUMEN
Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (M(W)=16.59 MDa and P(50)=41 mmHg) and relaxed (R) state PolyHb (M(W)=26.33 MDa and P(50)=0.66 mmHg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O(2) dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.