Liposomes with Low Levels of Grafted Poly(ethylene glycol) Remain Susceptible to Destabilization by Anti-Poly(ethylene glycol) Antibodies.

Binding of anti-PEG antibodies to poly(ethylene glycol) (PEG) on the surface of PEGylated liposomal doxorubicin (PLD) in vitro and in rats can activate complement and cause the rapid release of doxorubicin from the liposome interior. Here, we find that irinotecan liposomes (IL) and L-PLD, which have 16-fold lower levels of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 in their liposome membrane as compared to PLD, generate less complement activation but remain sensitive to destabilization and drug release by anti-PEG antibodies. Complement activation and liposome destabilization correlated with the theoretically estimated number of antibody molecules bound per liposome. Drug release from liposomes proceeded through the alternative complement pathway but was accelerated by the classical complement pathway. In contrast to PLD destabilization by anti-PEG immunoglobulin G (IgG), which proceeded by the insertion of membrane attack complexes in the lipid bilayer of otherwise intact PLD, anti-PEG IgG promoted the fusion of L-PLD, and IL to form unilamellar and oligo-vesicular liposomes. Anti-PEG immunoglobulin M (IgM) induced drug release from all liposomes (PLD, L-PLD, and IL) via the formation of unilamellar and oligo-vesicular liposomes. Anti-PEG IgG destabilized both PLD and L-PLD in rats, indicating that the reduction of PEG levels on liposomes is not an effective approach to prevent liposome destabilization by anti-PEG antibodies.

Engineering stable and non-immunogenic immunoenzymes for cancer therapy via in situ generated prodrugs.

Engineering human enzymes for therapeutic applications is attractive but introducing new amino acids may adversely affect enzyme stability and immunogenicity. Here we used a mammalian membrane-tethered screening system (ECSTASY) to evolve human lysosomal beta-glucuronidase (hBG) to hydrolyze a glucuronide metabolite (SN-38G) of the anticancer drug irinotecan (CPT-11). Three human beta-glucuronidase variants (hBG3, hBG10 and hBG19) with 3, 10 and 19 amino acid substitutions were identified that display up to 40-fold enhanced enzymatic activity, higher stability than E. coli beta-glucuronidase in human serum, and similar pharmacokinetics in mice as wild-type hBG. The hBG variants were two to three orders of magnitude less immunogenic than E. coli beta-glucuronidase in hBG transgenic mice. Intravenous administration of an immunoenzyme (hcc49-hBG10) targeting a sialyl-Tn tumor-associated antigen to mice bearing human colon xenografts significantly enhanced the anticancer activity of CPT-11 as measured by tumor suppression and mouse survival. Our results suggest that genetically-modified human enzymes represent a good alternative to microbially-derived enzymes for therapeutic applications.

Antiviral activity of Vigna radiata extract against feline coronavirus in vitro.

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

Pulmonary Hypertension Induces Serotonin Hyperreactivity and Metabolic Reprogramming in Coronary Arteries via NOX1/4-TRPM2 Signaling Pathway.

BACKGROUND: Clinical evidence revealed abnormal prevalence of coronary artery (CA) disease in patients with pulmonary hypertension (PH). The mechanistic connection between PH and CA disease is unclear. Serotonin (5-hydroxytryptamine), reactive oxygen species, and Ca2+ signaling have been implicated in both PH and CA disease. Our recent study indicates that NOXs (NADPH [nicotinamide adenine dinucleotide phosphate] oxidases) and TRPM2 (transient receptor potential cation channel subfamily M member 2) are key components of their interplay. We hypothesize that activation of the NOX-TRPM2 pathway facilitates the remodeling of CA in PH. METHODS: Left and right CAs from chronic hypoxia and monocrotaline-induced PH rats were collected to study vascular reactivity, gene expression, metabolism, and mitochondrial function. Inhibitors or specific siRNA were used to examine the pathological functions of NOX1/4-TRPM2 in CA smooth muscle cells. RESULTS: Significant CA remodeling and 5-hydroxytryptamine hyperreactivity in the right CA were observed in PH rats. NOX1/4-mediated reactive oxygen species production coupled with TRPM2-mediated Ca2+ influx contributed to 5-hydroxytryptamine hyperresponsiveness. CA smooth muscle cells from chronic hypoxia-PH rats exhibited increased proliferation, migration, apoptosis, and metabolic reprogramming in an NOX1/4-TRPM2-dependent manner. Furthermore, the NOX1/4-TRPM2 pathway participated in mitochondrial dysfunction, involving mitochondrial DNA damage, reactive oxygen species production, elevated mitochondrial membrane potential, mitochondrial Ca2+ accumulation, and mitochondrial fission. In vivo knockdown of NOX1/4 alleviated PH and suppressed CA remodeling in chronic hypoxia rats. CONCLUSIONS: PH triggers an increase in 5-hydroxytryptamine reactivity in the right CA and provokes metabolic reprogramming and mitochondrial disruption in CA smooth muscle cells via NOX1/4-TRPM2 activation. This signaling pathway may play an important role in CA remodeling and CA disease in PH.