RESUMO
Computational techniques are useful to predict interaction models and molecular properties for the design of drug delivery systems, such as dendrimers. This work evaluated the impact of surface modifications of mannosamine-conjugated multifunctional poly(glutamic acid) (PG)-dendrimers as nanocarriers of the tumour associated antigens (TAA) MART-1, gp100:44 and gp100:209. Molecular dynamics simulations and docking studies were performed. Nitrobenzoxadiazole (NBD)-PG-G4-dendrimer displayed 64 carboxylic groups, however, the Frontier Molecular Orbital Theory study evidenced that only 32 of those were available to form covalent bonds. When the number of mannosamines conjugated to dendrimer was increased from 16 to 32, the dendrimer interacted with the receptor with higher affinity. However, 16 mannosamines-NBD-PG-G4-dendrimer was chosen to conjugate TAA for added functionality as no carboxylic end groups were available for further conjugation in the 32 mannosamines-dendrimer. Docking results showed that the majority of TAA-conjugated NBD-PG-G4-dendrimer demonstrated a favourable interaction with mannosamine binding site on mannose receptor, thus constituting a promising tool for TAA targeted delivery. Our in silico approach effectively narrows down the selection of the best candidates for the synthesis of functionalised PG-dendrimers with desired functionalities. These results will significantly reduce the time and efforts required to experimentally synthesise modified dendrimers for optimal antigen delivery.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/química , Dendrímeros/química , Sistemas de Liberação de Medicamentos/tendências , Ácido Poliglutâmico/química , Vacinação/tendências , Sequência de Aminoácidos , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Computadores Moleculares/tendências , Dendrímeros/administração & dosagem , Dendrímeros/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Simulação de Acoplamento Molecular/tendências , Ácido Poliglutâmico/administração & dosagem , Ácido Poliglutâmico/genética , Ácido Poliglutâmico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Vacinação/métodosRESUMO
The Fc-fusion mimetic RpR 2[combining low line] was prepared by disulfide bridging conjugation using PEG in the place of the Fc. RpR 2[combining low line] displayed higher affinity for VEGF than aflibercept. This is caused primarily by a slower dissociation rate, which can prolong a drug at its site of action. RpRs have considerable potential for development as stable, organ specific therapeutics.
Assuntos
Receptores de Fatores de Crescimento do Endotélio Vascular/química , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Dissulfetos/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Peso Molecular , Polietilenoglicóis/química , Ligação Proteica/imunologia , Multimerização ProteicaRESUMO
PURPOSE: To compare and examine the storage stability of compounded bevacizumab in polycarbonate (PC) and polypropylene (PP) syringes over a 6-month period. PC syringes have been used in a recent clinical study and bevacizumab stability has not been reported for this type of syringe. METHODS: Repackaged bevacizumab was obtained from Moorfields Pharmaceuticals in PC and PP syringes. Bevacizumab from the stored syringes was analysed at monthly time points for a 6-month period and compared with bevacizumab from a freshly opened vial at each time point. SDS-PAGE electrophoresis and size-exclusion chromatography (SEC) was used to observe aggregation and degradation. Dynamic light scattering (DLS) provided information about the hydrodynamic size and particle size distribution of bevacizumab in solution. VEGF binding and the active concentration of bevacizumab was determined by surface plasmon resonance (SPR) using Biacore. RESULTS: SDS-PAGE and SEC analysis did not show any changes in the presence of higher molecular weight species (HMWS) or degradation products in PC and PP syringes from T0 to T6 compared with bevacizumab sampled from a freshly opened vial. The hydrodynamic diameter of bevacizumab in the PC syringe after 6 months of storage was not significantly different to bevacizumab taken from a freshly opened vial. Using SPR, the VEGF binding activity of bevacizumab in the PC syringe was comparable to bevacizumab taken from a freshly opened vial. CONCLUSION: No significant difference over a 6-month period was observed in the quality of bevacizumab repackaged into prefilled polycarbonate and polypropylene syringes when compared with bevacizumab that is supplied from the vial.
Assuntos
Inibidores da Angiogênese/análise , Bevacizumab/análise , Polímeros , Polipropilenos , Seringas , Inibidores da Angiogênese/química , Bevacizumab/química , Composição de Medicamentos , Embalagem de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Humanos , Polímeros/química , Polipropilenos/química , Multimerização Proteica , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.
Assuntos
Acrilamidas/farmacocinética , Antineoplásicos Fitogênicos/farmacocinética , Elipticinas/farmacocinética , Polímeros/farmacocinética , Acrilamidas/administração & dosagem , Acrilamidas/síntese química , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/síntese química , Divisão Celular/efeitos dos fármacos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/síntese química , Portadores de Fármacos/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Elipticinas/administração & dosagem , Elipticinas/síntese química , Hemólise/efeitos dos fármacos , Masculino , Metacrilatos/administração & dosagem , Metacrilatos/síntese química , Metacrilatos/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Polímeros/administração & dosagem , Polímeros/síntese química , Solubilidade , Equivalência Terapêutica , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/transplanteRESUMO
The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.