Pharmacokinetics of protein and peptide conjugates

Protein and peptide conjugates have become an important component of therapeutic and diagnostic medicine. These conjugates are primarily designed to improve pharmacokinetics (PK) of those therapeutic or imaging agents, which do not possess optimal disposition characteristics. In this review we have summarized preclinical and clinical PK of diverse protein and peptide conjugates, and have showcased how different conjugation approaches are used to obtain the desired PK. We have classified the conjugates into peptide conjugates, non-targeted protein conjugates, and targeted protein conjugates, and have highlighted diagnostic and therapeutic applications of these conjugates. In general, peptide conjugates demonstrate very short half-life and rapid renal elimination, and they are mainly designed to achieve high contrast ratio for imaging agents or to deliver therapeutic agents at sites not reachable by bulky or non-targeted proteins. Conjugates made from non-targeted proteins like albumin are designed to increase the half-life of rapidly eliminating therapeutic or imaging agents, and improve their delivery to tissues like solid tumors and inflamed joints. Targeted protein conjugates are mainly developed from antibodies, antibody derivatives, or endogenous proteins, and they are designed to improve the contrast ratio of imaging agents or therapeutic index of therapeutic agents, by enhancing their delivery to the site-of-action.     

Multicompartment systems: A putative carrier for combined drug delivery and targeting

In this review, we discuss recent developments in multicompartment systems commonly referred to as vesosomes, as well as their method of preparation, surface modifications, and clinical potential. Vesosomal systems are able to entrap more than one drug moiety and can be customized for site-specific delivery. We focus in particular on the possible reticuloendothelial system (RES) – mediated accumulation of vesosomes, and their application in tumor targeting, as areas for further investigation.     

Pegylated nanoceria: A versatile nanomaterial for noninvasive treatment of retinal diseases

Oxidative stress induced reactive oxygen species has been implicated as the primary molecular mechanism in the pathogenesis of debilitating retinal diseases such as diabetic retinopathy, neovascularization and age-related macular degeneration. Nanoceria (cerium oxide nanoparticles) has recently received much attention, because of its superior and regenerative radical scavenging properties. This review focuses on retinal applications of nanoceria and functionalized nanoceria. Studies in animal models showed that nanoceria possess antioxidant, anti-inflammatory, anti-angiogenic, anti-apoptotic properties and preserves retinal morphology and prevents loss of retinal functions. Nanoceria have been tested in animal models of age-related macular degeneration and neovascularization and their efficacy have been shown to persist for a long time, without any collateral effects. To date, several pharmaceutical formulations of nanoceria have been developed for their prospective clinical ophthalmic applications such as chitosan coated nanoceria, nanoceria loaded into hydrogels, nanoceria embedded in wafers and contact lens and organosilane or polyethylene glycol functionalized nanoceria. Based on their nano size range, ocular permeation could be achieved to allow topical administration of nanoceria. PEGylation of nanoceria represents the key strategy to support eye drop formulation with enhanced corneal permeation, without altering chemical physical properties. Based on their excellent antioxidant properties, nano-size, safety and tolerability, PEGylated nanoceria represent a new potential therapeutic for the treatment.     

多模微波辅助的聚乙二醇化聚苯乙烯树脂制备

目的：研究一种微波辅助的聚苯乙烯树脂聚乙二醇（PEG）化的方法。方法使用PEG 200对市售Merrifield树脂进行修饰,尝试了不同条件下PEG化的效率。使用自行制备的PEG化树脂进一步衍生化,并进行氨基酸的负载,与市售王树脂进行比对。结果优选出了一套较为合适的反应参数,使用5 g树脂,50 ml PEG 200,微波功率600 W,预设温度170℃,照射15 min,共2次,获得了接近90%的负载率。衍生化得到PEG化王树脂后与氨基酸进行缩合,获得了令人满意的产率。结论本文首次报道了多模微波装置辅助的聚苯乙烯树脂PEG化,该方法操作简单,制备快速,条件温和,产率满意,远远优于传统制备方法。     

Universal Bioinert Control of Polystyrene Interfaces via Hydrophobic‐Driven Self‐Assembled Surface PEGylation with a Well‐Defined Block Sequence

This work proposes a new molecular insight of interfacial design in the control of antifouling performance for the versatile biofoulants, including proteins, blood cells, tissue cells, and bacteria. A self‐assembled bioinert interface with universal fouling resistance to general biofoulants via hydrophobic‐driven surface PEGylation is presented. The study systematically discriminates the optimum PEGylated block polymer configuration and hydrophobic/hydrophilic segmental ratio enabling to optimize the surface coverage by the bioinert moieties, thus ensuring the best resistance to biofouling. For similar copolymer molecular weights and similar polystyrene (PS)/poly(ethylene glycol) methacrylate (PEGMA), the coating density obtainable is the highest if a random copolymer is used, while it is the lowest with a triblock copolymer. That measured with a diblock copolymer lies in between. Random copolymers offer more numerous anchoring possibilities than diblock copolymers, while they are importantly fewer if triblock copolymers are used. For similar total number of hydrophilic blocks, the diblock copolymer is more efficient to resist larger cells (leukocytes, fibroblasts) while the triblock is better to promote mitigate biofouling by smaller molecules or cells (proteins, platelets, red blood cells). The length of the hydrophilic PEGylated block seems to dominate fouling resistance of large biofoulants.     

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔG_unf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.     

Benefits of PEGylation in the early post-transplant period of intraportal islet transplantation as assessed by magnetic resonance imaging of labeled islets

While a few studies have demonstrated the benefit of PEGylation in islet transplantation, most have employed renal subcapsular models and none have performed direct comparisons of islet mass in intraportal islet transplantation using islet magnetic resonance imaging (MRI). In this study, our aim was to demonstrate the benefit of PEGylation in the early post-transplant period of intraportal islet transplantation with a novel algorithm for islet MRI. Islets were PEGylated after ferucarbotran labeling in a rat syngeneic intraportal islet transplantation model followed by comparisons of post-transplant glycemic levels in recipient rats infused with PEGylated (n = 12) and non-PEGylated (n = 13) islets. The total area of hypointense spots and the number of hypointense spots larger than 1.758 mm² of PEGylated and non-PEGylated islets were quantitatively compared. The total area of hypointense spots (P < 0.05) and the number of hypointense spots larger than 1.758 mm² (P < 0.05) were higher in the PEGylated islet group 7 and 14 days post translation (DPT). These results translated into better post-transplant outcomes in the PEGylated islet group 28 DPT. In validation experiments, MRI parameters obtained 1, 7, and 14 DPT predicted normoglycemia 4 wk post-transplantation. We directly demonstrated the benefit of islet PEGylation in protection against nonspecific islet destruction in the early post-transplant period of intraportal islet transplantation using a novel algorithm for islet MRI. This novel algorithm could serve as a useful tool to demonstrate such benefit in future clinical trials of islet transplantation using PEGylated islets.     

Poly(ethylene glycol)-block-cationic polylactide nanocomplexes of differing charge density for gene delivery

Representing a new type of biodegradable cationic block copolymer, well-defined poly(ethylene glycol)-block-cationic polylactides (PEG-b-CPLAs) with tertiary amine-based cationic groups were synthesized by thiol-ene functionalization of an allyl-functionalized diblock precursor. Subsequently the application of PEG-b-CPLAs as biodegradable vectors for the delivery of plasmid DNAs (pDNAs) was investigated. Via the formation of PEG-b-CPLA:pDNA nanocomplexes by spontaneous electrostatic interaction, pDNAs encoding luciferase or enhanced green fluorescent protein were successfully delivered to four physiologically distinct cell lines (including macrophage, fibroblast, epithelial, and stem cell). Formulated nanocomplexes demonstrated high levels of transfection with low levels of cytotoxicity and hemolysis when compared to a positive control. Biophysical characterization of charge densities of nanocomplexes at various polymer:pDNA weight ratios revealed a positive correlation between surface charge and gene delivery. Nanocomplexes with high surface charge densities were utilized in an in vitro serum gene delivery inhibition assay, and effective gene delivery was observed despite high levels of serum. Overall, these results help to elucidate the influence of charge, size, and PEGylation of nanocomplexes upon the delivery of nucleic acids in physiologically relevant conditions.     

Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent that specifically induces apoptosis in broad-spectrum tumor cell lines, meanwhile leaving normal cells unaffected. Unfortunately, the clinical development of TRAIL was hampered, and could be attributed to its instability, bioavailability or poor delivery. Although N-terminal specific PEGylation provides a means to improve the pharmacokinetic and stability of TRAIL, it took a bit longer time to accomplish the PEGylation process than expected. We therefore designed another PEGylation approach, mutated Cys-SH site-specific PEGylation, to conjugate methoxypoly(ethylene glycol) maleimide (mPEG-MAL) with TRAIL (95–281) mutant N109C. Asn-109 was chosen as the PEGylated site for it is a potential N-linked glycosylation site. It was shown that ∼90% TRAIL mutant N109C could be PEGylated by mPEG-MAL within 40 min. And mPEG_MAL-N109C was revealed to possess superior in vitro stability and antitumor activity than N-terminal specifically PEGylated TRAIL (114–281) (mPEG_ALD-TRAIL_114–281). What's more, mPEG_MAL-N109C exhibited more therapeutic potentials than mPEG_ALD-TRAIL_114–281 in tumor xenograft model, benefitting from better drug delivery and bioavailability. These results have demonstrated mutated Cys-SH specific PEGylation is an alternative to site-specifically PEGylate TRAIL efficiently and effectively other than N-terminal specific PEGylation.     

聚乙二醇化Exendin-4类似物受体亲和力测定方法的建立

基于荧光素酶报告基因法和本实验室前期构建的CHO-GLP-1R-CRE-Luc⁺检测模型细胞株,建立并优化了长效降糖多肽——聚乙二醇化Exendin-4类似物PE与GLP-1受体的亲和力测定方法,并对其进行了方法学验证。结果表明,当药物作用4 h,化学发光底物作用15 min,检测药物在5.7×10^-3~1.5×10³ nmol/L浓度区间内时,该检测方法的专属性和耐用性良好、准确度和精密度较高,符合《生物制品质量控制分析方法验证技术一般原则》的相关要求。本研究为该类GLP-1受体激动剂药物的快速评价与筛选奠定了基础。