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1.
Langmuir ; 40(13): 6685-6693, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38525517

RESUMO

Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of ß-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.


Assuntos
NAD , NAD/química , Biocatálise , Catálise , Cinética , Adsorção
2.
Mol Pharm ; 20(6): 3073-3087, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37218930

RESUMO

Covalent conjugation of a biologically stable polymer to a therapeutic protein, e.g., an antibody, holds many benefits such as prolonged plasma exposure of the protein and improved tumor uptake. Generation of defined conjugates is advantageous in many applications, and a range of site-selective conjugation methods have been reported. Many current coupling methods lead to dispersity in coupling efficiencies with subsequent conjugates of less-well-defined structure, which impacts reproducibility of manufacture and ultimately may impact successful translation to treat or image diseases. We explored designing stable, reactive groups for polymer conjugation reactions that would lead to conjugates through the simplest and most abundant residue on most proteins, the lysine residue, yielding conjugates in high purity and demonstrating retention of mAb efficacy through surface plasmon resonance (SPR), cell targeting, and in vivo tumor targeting. We utilized squaric acid diesters as coupling agents for selective amidation of lysine residues and were able to selectively conjugate one, or two, high-molecular-weight polymers to a therapeutically relevant antibody, 528mAb, that subsequently retained full binding specificity. Water-soluble copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) and N-isopropylacrylamide (NIPAM) were prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) polymerization and we demonstrated that a dual-dye-labeled antibody-RAFT conjugate (528mAb-RAFT) exhibited effective tumor targeting in model breast cancer xenografts in mice. The combination of the precise and selective squaric acid ester conjugation method, with the use of RAFT polymers, leads to a promising strategic partnership for improved therapeutic protein-polymer conjugates having a very-well-defined structure.


Assuntos
Neoplasias , Polímeros , Humanos , Animais , Camundongos , Polímeros/química , Lisina , Reprodutibilidade dos Testes , Anticorpos , Proteínas/química
3.
Mol Pharm ; 18(9): 3464-3474, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34448393

RESUMO

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Moléculas de Adesão Celular/antagonistas & inibidores , Corantes Fluorescentes/administração & dosagem , Imagem Óptica/métodos , Neoplasias Ovarianas/diagnóstico , Animais , Anticorpos Monoclonais/química , Antígenos de Neoplasias , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Humanos , Verde de Indocianina/administração & dosagem , Verde de Indocianina/química , Injeções Intravenosas , Camundongos , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Bioconjug Chem ; 30(10): 2539-2543, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31560523

RESUMO

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Sítios de Ligação , Corantes Fluorescentes/química , Células HEK293 , Humanos , Ligantes , Coloração e Rotulagem
5.
Biomacromolecules ; 18(9): 2866-2875, 2017 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-28731677

RESUMO

Interferon α2 is an antiviral/antiproliferative protein that is currently used to treat hepatitis C infections and several forms of cancer. Two PEGylated variants of interferon α2 (containing 12 and 40 kDa PEGs) are currently marketed and display longer plasma circulation times than that of unmodified interferon. With increasing realization that the lymphatic system plays an important role in the extrahepatic replication of the hepatitis C virus and in the metastatic dissemination of cancers, this study sought to evaluate PEGylation strategies to optimally enhance the antiviral activity and plasma and lymphatic exposure of interferon after subcutaneous administration in rats. The results showed that conjugation with a linear 20 kDa PEG provided the most ideal balance between activity and plasma and lymph exposure. A linear 5 kDa PEG variant also exhibited excellent plasma and lymph exposure to interferon activity when compared to those of unmodified interferon and the clinically available linear 12 kDa PEGylated construct.


Assuntos
Antivirais/síntese química , Interferon-alfa/síntese química , Sistema Linfático/metabolismo , Polietilenoglicóis/síntese química , Animais , Antivirais/administração & dosagem , Antivirais/química , Antivirais/farmacocinética , Injeções Subcutâneas , Interferon-alfa/administração & dosagem , Interferon-alfa/química , Interferon-alfa/farmacocinética , Masculino , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Distribuição Tecidual
6.
Anal Chem ; 88(20): 10102-10110, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27644116

RESUMO

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

7.
Mol Pharm ; 13(4): 1229-41, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-26871003

RESUMO

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Polietilenoglicóis/química , Trastuzumab/imunologia , Animais , Linhagem Celular Tumoral , Cromatografia em Gel , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
8.
Mol Pharm ; 12(3): 794-809, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25644368

RESUMO

The lymphatic system plays a major role in the metastatic dissemination of cancer and has an integral role in immunity. PEGylation enhances drainage and lymphatic uptake following subcutaneous (sc) administration of proteins and protein-like polymers, but the impact of PEGylation of very large proteins (such as antibodies) on subcutaneous and lymphatic pharmacokinetics is unknown. This study therefore aimed to evaluate the impact of PEGylation on the sc absorption and lymphatic disposition of the anti-HER2 antibody trastuzumab in rats. PEG-trastuzumab was generated via the conjugation of a single 40 kDa PEG-NHS ester to trastuzumab. PEG-trastuzumab showed a 5-fold reduction in HER2 binding affinity, however the in vitro growth inhibitory effects were preserved as a result of changes in cellular trafficking when compared to native trastuzumab. The lymphatic pharmacokinetics of PEG-trastuzumab was evaluated in thoracic lymph duct cannulated rats after iv and sc administration and compared to the pharmacokinetics of native trastuzumab. The iv pharmacokinetics and lymphatic exposure of PEG-trastuzumab was similar when compared to trastuzumab. After sc administration, initial plasma pharmacokinetics and lymphatic exposure were also similar between PEG-trastuzumab and trastuzumab, but the absolute bioavailability of PEG-trastuzumab was 100% when compared to 86.1% bioavailability for trastuzumab. In contrast to trastuzumab, PEG-trastuzumab showed accelerated plasma clearance beginning approximately 7 days after sc, but not iv, administration, presumably as a result of the generation of anti-PEG IgM. This work suggests that PEGylation does not significantly alter the lymphatic disposition of very large proteins, and further suggests that it is unlikely to benefit therapy with monoclonal antibodies.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Trastuzumab/administração & dosagem , Trastuzumab/metabolismo , Administração Intravenosa , Animais , Antineoplásicos/química , Biofarmácia , Permeabilidade Capilar , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Injeções Subcutâneas , Linfa/metabolismo , Sistema Linfático/metabolismo , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , Trastuzumab/química
9.
Org Biomol Chem ; 12(17): 2675-85, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24643508

RESUMO

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aß, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aß-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.


Assuntos
Aminoaciltransferases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Embrião de Mamíferos/metabolismo , Neurônios/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Etilenodiaminas/metabolismo , Imunofluorescência , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Oligopeptídeos/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/metabolismo , Espectrometria de Massas por Ionização por Electrospray
10.
Angew Chem Int Ed Engl ; 53(24): 6115-9, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24777818

RESUMO

The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Complexos de Coordenação/química , Cobre/química , Cisteína Endopeptidases/química , Anticorpos de Cadeia Única/química , Animais , Camundongos , Estrutura Molecular
11.
Nat Nanotechnol ; 19(4): 534-544, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38168926

RESUMO

Injectable insulin is an extensively used medication with potential life-threatening hypoglycaemic events. Here we report on insulin-conjugated silver sulfide quantum dots coated with a chitosan/glucose polymer to produce a responsive oral insulin nanoformulation. This formulation is pH responsive, is insoluble in acidic environments and shows increased absorption in human duodenum explants and Caenorhabditis elegans at neutral pH. The formulation is sensitive to glucosidase enzymes to trigger insulin release. It is found that the formulation distributes to the liver in mice and rats after oral administration and promotes a dose-dependent reduction in blood glucose without promoting hypoglycaemia or weight gain in diabetic rodents. Non-diabetic baboons also show a dose-dependent reduction in blood glucose. No biochemical or haematological toxicity or adverse events were observed in mice, rats and non-human primates. The formulation demonstrates the potential to orally control blood glucose without hypoglycaemic episodes.


Assuntos
Hipoglicemia , Insulina , Ratos , Camundongos , Animais , Glicemia , Hipoglicemia/tratamento farmacológico , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/efeitos adversos
12.
Photochem Photobiol Sci ; 12(6): 1086-90, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23588201

RESUMO

Previous reports of unusual dual fluorescence in several porphyrazines proposed for use in photodynamic therapy and biomedical imaging have been re-examined. The blue-violet emissions previously assigned to S2-S0 fluorescence of these porphyrazines are shown to exhibit fluorescence excitation spectra that do not coincide with their absorption spectra, and the photophysical properties of the emission calculated from measured quantum yields and decay parameters do not match those calculated from the absorption spectra. In addition, the blue-violet emission intensities increase on exposure of aerated solutions of the porphyrazines to a broad spectrum of UV-visible light. The blue-violet emissions previously assigned to S2-S0 fluorescence of these porphyrazines themselves are therefore re-assigned to S1-S0 fluorescence of small amounts of photolysis products strongly absorbing in the same near UV spectral regions.


Assuntos
Porfirinas/química , Fotólise , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
13.
Biotechnol Bioeng ; 109(6): 1461-70, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22170409

RESUMO

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Receptores ErbB/antagonistas & inibidores , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem/métodos , Aminoaciltransferases/genética , Anticorpos Bloqueadores/genética , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/metabolismo , Proteínas de Bactérias/genética , Biotina/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Receptores ErbB/imunologia , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
14.
Chem Sci ; 12(26): 9004-9016, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34276928

RESUMO

Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (89Zr, 64Cu) into an anti-EGFR antibody fragment for Positron Emission Tomography (PET) imaging the same day as injection. A monovalent antibody fragment with high affinity for EGFR was engineered to include a sequence that is recognized by the transpeptidase sortase A. Two different metal chelators, one for 89ZrIV and one for 64CuII, were modified with a N-terminal glycine to enable them to act as substrates in sortase A mediated bioconjugation to the antibody fragment. Both fragments provided high-quality PET images of EGFR positive tumors in a mouse model at 3 hours post-injection, a significant advantage when compared to radiolabeled full antibodies that require several days between injection of the tracer and imaging. The use of enzymatic bioconjugation gives reproducible homogeneous products with the metal complexes selectively installed on the C-terminus of the antibody potentially simplifying regulatory approval.

15.
Int J Pharm ; 608: 121075, 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34481889

RESUMO

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.


Assuntos
Polietilenoglicóis , Polímeros , Animais , Metacrilatos , Ratos , Trastuzumab
16.
Eur J Pharm Biopharm ; 137: 218-226, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30851352

RESUMO

HIV therapy with anti-retroviral drugs is limited by the poor exposure of viral reservoirs, such as lymphoid tissue, to these small molecule drugs. We therefore investigated the effect of PEGylation on the anti-retroviral activity and subcutaneous lymphatic pharmacokinetics of the peptide-based fusion inhibitor enfuvirtide in thoracic lymph duct cannulated rats. Both the peptide and the PEG were quantified in plasma and lymph via ELISA. Conjugation to a single 5 kDa linear PEG decreased anti-HIV activity three-fold compared to enfuvirtide. Whilst plasma and lymphatic exposure to peptide mass was moderately increased, the loss of anti-viral activity led to an overall decrease in exposure to enfuvirtide activity. A 20 kDa 4-arm branched PEG conjugated with an average of two enfuvirtide peptides decreased peptide activity by six-fold. Plasma and lymph exposure to enfuvirtide, however, increased significantly such that anti-viral activity was increased two- and six-fold respectively. The results suggest that a multi-enfuvirtide-PEG complex may optimally enhance the anti-retroviral activity of the peptide in plasma and lymph.


Assuntos
Enfuvirtida/administração & dosagem , Inibidores da Fusão de HIV/administração & dosagem , HIV/efeitos dos fármacos , Polietilenoglicóis/química , Animais , Linhagem Celular , Enfuvirtida/farmacocinética , Enfuvirtida/farmacologia , Ensaio de Imunoadsorção Enzimática , Inibidores da Fusão de HIV/farmacocinética , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Humanos , Linfa/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
17.
Chem Commun (Camb) ; (25): 2897-9, 2008 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-18566717

RESUMO

The crystal structure of a cyanine dye rotaxane shows that the cyclodextrin is tightly threaded round the polymethine bridge of the dye; encapsulation dramatically increases the kinetic chemical stability of the radicals formed on oxidation and reduction of the dye, making it possible to observe the rotaxane radical dication by ESR and UV-vis-NIR spectroscopy.

18.
J Org Chem ; 73(23): 9340-52, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18983188

RESUMO

This paper describes the synthesis, structural characterization, and solution behavior of some xylyl-linked imidazolium and benzimidazolium cyclophanes decorated with alkyl or alkoxy groups. The addition of alkyl/alkoxy chains to the cyclophanes allows for studies in chlorinated solvents, whereas previous solution studies of azolium cyclophanes have generally required highly polar solvents. The azolium cyclophanes may exist in a syn/syn conformation (azolium rings mutually syn, arene rings mutually syn) or a syn/anti conformation (azolium rings mutually syn, arene rings mutually anti). The preferred conformation is significantly affected by (i) binding of bromide (ion pairing) to the protons on the imidazolium or benzimidazolium rings, which occurs in solutions of bromide salts of the cyclophanes in chlorinated solvents, and (ii) the addition of alkoxy groups to the benzimidazolium cyclophanes. These structural modifications have also led to cyclophanes that adopt conformations not previously identified for similar azolium cyclophane analogues. Detailed (1)H NMR studies for one cyclophane identified binding of bromide at two independent sites within the cyclophane.

19.
PLoS One ; 12(11): e0184183, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29112947

RESUMO

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.


Assuntos
Acetato Quinase/metabolismo , Catalase/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Glicerol Quinase/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Açúcares/síntese química , Biocatálise , Cromatografia Líquida de Alta Pressão , Estereoisomerismo , Açúcares/química
20.
Drug Deliv ; 24(1): 1770-1781, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29160134

RESUMO

The advent of nanomedicine requires novel delivery vehicles to actively target their site of action. Here, we demonstrate the development of lung-targeting drug-loaded liposomes and their efficacy, specificity and safety. Our study focuses on glucocorticoids methylprednisolone (MPS), a commonly used drug to treat lung injuries. The steroidal molecule was loaded into functionalized nano-sterically stabilized unilamellar liposomes (NSSLs). Targeting functionality was performed through conjugation of surfactant protein A (SPANb) nanobodies to form MPS-NSSLs-SPANb. MPS-NSSLs-SPANb exhibited good size distribution, morphology, and encapsulation efficiency. Animal experiments demonstrated the high specificity of MPS-NSSLs-SPANb to the lung. Treatment with MPS-NSSLs-SPANb reduced the levels of TNF-α, IL-8, and TGF-ß1 in rat bronchoalveolar lavage fluid and the expression of NK-κB in the lung tissues, thereby alleviating lung injuries and increasing rat survival. The nanobody functionalized nanoparticles demonstrate superior performance to treat lung injury when compared to that of antibody functionalized systems.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lipossomos/química , Metilprednisolona/química , Metilprednisolona/farmacologia , Nanopartículas/química , Proteína A Associada a Surfactante Pulmonar/química , Animais , Líquido da Lavagem Broncoalveolar/química , Sistemas de Liberação de Medicamentos/métodos , Glucocorticoides/química , Glucocorticoides/farmacologia , Interleucina-8/metabolismo , Pulmão/efeitos dos fármacos , Masculino , Surfactantes Pulmonares/química , Surfactantes Pulmonares/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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