Downregulation of Interferon-γ Receptor Expression Endows Resistance to Anti-Programmed Death Protein 1 Therapy in Colorectal Cancer.

Immune checkpoint inhibitors have emerged as a frontline treatment of a variety of malignancies. However, only a subset of patients respond to these therapies, and many initial responders eventually develop resistance, leading to tumor relapse. Programmed death protein 1 is one of the checkpoint inhibitors that is expressed on activated T cells and suppresses the antitumor immune response when binding to its ligand, programmed death ligand 1, on tumor cells. Previous studies indicated that loss-of-function mutations in the IFN-γ pathway could result in acquired resistance to immune checkpoint inhibitors in human patients with cancer. Here, we investigated the effects of the IFN-γ receptor downexpression on the response to an anti-PD-1 antibody (αPD1) in a murine colorectal cancer model and the underlying mechanisms of resistance. IFN-γ receptor (IFNGR) 1 was knocked down in MC38 cells, a murine colon adenocarcinoma cell line using IFNGR1 short hairpin RNA (shRNA) lentiviral particles. Then, MC38 IFNGR1 knockdown (KD) cells and negative control (SC) cells were used in this study. In the C57BL/6 xenograft model, the KD tumor demonstrated resistance to αPD1 in comparison with SC cells. The observed treatment resistance might be associated with reduced tumor-infiltrating immune cells (TILs). When mixed, the resistant (KD) and control cells (SC) grew in spatially separated tumor areas, and αPD1 did not impact this pattern of spatial distribution. Our findings have proved that downregulation of the IFNGR1 endowed resistance to αPD1 and provided the potential mechanisms involving the TILs. SIGNIFICANCE STATEMENT: Immunological checkpoint blockades have achieved substantial efficacy in a variety of tumors. However, only a subset of patients respond to these therapies, and innate and acquired resistance is widely present. Our study found that the downregulation of the IFN-γ receptor caused resistance to an anti-PD-1 antibody in a murine colorectal cancer model associated with the reduced tumor-infiltrating lymphocytes. Our findings have substantial implications for improving the efficacy of checkpoint blockades.

Correction to "Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer".

[This corrects the article PMC10317193.].

A systems pharmacokinetic/pharmacodynamic model for concizumab to explore the potential of anti-TFPI recycling antibodies.

Concizumab is a humanized monoclonal antibody in clinical investigation directed against membrane-bound and soluble tissue factor pathway inhibitor (mTFPI and sTFPI) for treatment of hemophilia. Concizumab displays a non-linear pharmacokinetic (PK) profile due to mTFPI-mediated endocytosis and necessitates a high dose and frequent dosing to suppress the abundant sTFPI, a negative regulator of coagulation. Recycling antibodies that can dissociate bound mTFPI/sTFPI in endosomes for degradation and rescue antibody from degradation have a potential in reducing the dose by extending antibody systemic persistence and sTFPI suppression. We developed a systems PK/pharmacodynamics (PD) model with nested endosome compartments to simulate the effect of decreased antibody binding to mTFPI/sTFPI in endosomes on antibody clearance and sTFPI suppression for exploring the potential of anti-TFPI recycling antibodies in reducing the dose. A dynamic model-building strategy was taken. A reduced PK/PD model without the endosome compartments was developed to optimize unknown target turnover parameters using concizumab PK data. The optimized parameters were then employed in the systems PK/PD model for simulations. The obtained systems PK/PD model adequately described the PK of concizumab in rabbits, monkeys, and humans and the PD in humans. The systems PK/PD model predicted that an anti-TFPI recycling antibody with a 100-fold higher mTFPI/sTFPI dissociation constant in endosomes than concizumab can extend sTFPI suppression from 12 days to 1 month. Thus, the systems PK/PD model provides a quantitative platform for guiding the engineering and translational development of anti-TFPI recycling antibodies.

Pharmacokinetics and Pharmacodynamics Modeling and Simulation Systems to Support the Development and Regulation of Liposomal Drugs.

Liposomal formulations have been developed to improve the therapeutic index of encapsulated drugs by altering the balance of on- and off-targeted distribution. The improved therapeutic efficacy of liposomal drugs is primarily attributed to enhanced distribution at the sites of action. The targeted distribution of liposomal drugs depends not only on the physicochemical properties of the liposomes, but also on multiple components of the biological system. Pharmacokinetic⁻pharmacodynamic (PK⁻PD) modeling has recently emerged as a useful tool with which to assess the impact of formulation- and system-specific factors on the targeted disposition and therapeutic efficacy of liposomal drugs. The use of PK⁻PD modeling to facilitate the development and regulatory reviews of generic versions of liposomal drugs recently drew the attention of the U.S. Food and Drug Administration. The present review summarizes the physiological factors that affect the targeted delivery of liposomal drugs, challenges that influence the development and regulation of liposomal drugs, and the application of PK⁻PD modeling and simulation systems to address these challenges.

Physiologically Based Pharmacokinetic Modeling of Nanoparticles.

Nanoparticles are frequently designed to improve the pharmacokinetics profiles and tissue distribution of small molecules to prolong their systemic circulation, target specific tissue, or widen the therapeutic window. The multifunctionality of nanoparticles is frequently presented as an advantage but also results in distinct and complicated in vivo disposition properties compared with a conventional formulation of the same molecules. Physiologically based pharmacokinetic (PBPK) modeling has been a useful tool in characterizing and predicting the systemic disposition, target exposure, and efficacy and toxicity of various types of drugs when coupled with pharmacodynamic modeling. Here we review the unique disposition characteristics of nanoparticles, assess how PBPK modeling takes into account the unique disposition properties of nanoparticles, and comment on the applications and challenges of PBPK modeling in characterizing and predicting the disposition and biological effects of nanoparticles.

A Minimal Physiologically Based Pharmacokinetic Model with a Nested Endosome Compartment for Novel Engineered Antibodies.

We proposed here a minimal physiologically based pharmacokinetic (mPBPK) model for a group of novel engineered antibodies in mice and humans. These antibodies are designed with altered binding properties of their Fc domain with neonatal Fc receptor (FcRn) or the Fab domain with their cognate targets (recycling antibodies) in acidic endosomes. To enable simulations of such binding features in the change of antibody pharmacokinetics and its target suppression, we nested an endothelial endosome compartment in parallel with plasma compartment based on our previously established mPBPK model. The fluid-phase pinocytosis rate from plasma to endothelial endosomes was reflected by the clearance of antibodies in FcRn dysfunctional humans or FcRn-knockout mice. The endosomal recycling rate of FcRn-bound antibodies was calculated based on the reported endosomal transit time. The nonspecific catabolism in endosomes was fitted using pharmacokinetic data of a human wild-type IgG1 adalimumab in humans and B21M in human FcRn (hFcRn) transgenic mice. The developed model adequately predicted the pharmacokinetics of infliximab, motavizumab, and an Fc variant of motavizumab in humans and the pharmacokinetics of bevacizumab, an Fc variant of bevacizumab, and a recycling antibody PH-IgG1 and its non-pH dependent counterpart NPH-IgG1 in hFcRn transgenic mice. Our proposed model provides a platform for evaluation of the pharmacokinetics and disposition behaviors of Fc-engineered antibodies and recycling antibodies.

Engineering macrophage-derived exosomes for targeted paclitaxel delivery to pulmonary metastases: in vitro and in vivo evaluations.

Exosomes have recently emerged as a promising drug delivery system with low immunogenicity, high biocompatibility, and high efficacy of delivery. We demonstrated earlier that macrophage-derived exosomes (exo) loaded with a potent anticancer agent paclitaxel (PTX) represent a novel nanoformulation (exoPTX) that shows high anticancer efficacy in a mouse model of pulmonary metastases. We now report the manufacture of targeted exosome-based formulations with superior structure and therapeutic indices for systemic administration. Herein, we developed and optimized a formulation of PTX-loaded exosomes with incorporated aminoethylanisamide-polyethylene glycol (AA-PEG) vector moiety to target the sigma receptor, which is overexpressed by lung cancer cells. The AA-PEG-vectorized exosomes loaded with PTX (AA-PEG-exoPTX) possessed a high loading capacity, profound ability to accumulate in cancer cells upon systemic administration, and improved therapeutic outcomes. The combination of targeting ability with the biocompatibility of exosome-based drug formulations offers a powerful and novel delivery platform for anticancer therapy.

Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain.

Recent work has stimulated interest in the use of exosomes as nanocarriers for delivery of small drugs, RNAs, and proteins to the central nervous system (CNS). To overcome the blood-brain barrier (BBB), exosomes were modified with brain homing peptides that target brain endothelium but likely to increase immune response. Here for the first time we demonstrate that there is no need for such modification to penetrate the BBB in mammals. The naïve macrophage (Mϕ) exosomes can utilize, 1) on the one hand, the integrin lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), and, 2) on the other hand, the carbohydrate-binding C-type lectin receptors, to interact with brain microvessel endothelial cells comprising the BBB. Notably, upregulation of ICAM-1, a common process in inflammation, promotes Mϕ exosomes uptake in the BBB cells. We further demonstrate in vivo that naïve Mϕ exosomes, after intravenous (IV) administration, cross the BBB and deliver a cargo protein, the brain derived neurotrophic factor (BDNF), to the brain. This delivery is enhanced in the presence of brain inflammation, a condition often present in CNS diseases. Taken together, the findings are of interest to basic science and possible use of Mϕ-derived exosomes as nanocarriers for brain delivery of therapeutic proteins to treat CNS diseases.

Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity.

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

Effect of ligand density and PEG modification on octreotide-targeted liposome via somatostatin receptor in vitro and in vivo.

Octreotide had been exploited as a targeting ligand for nanoparticle tumor localization overexpressing somatostatin receptor. In addition to particle size and other physiochemical properties, ligand density had great influence on the delivery of active targeted nanoparticles. Herein, octreotide-targeted liposomal doxorubicin was constructed with different ligand density by post-inserting HSPE-PEG4000-Octreotide into pre-formed liposome. The octreotide ligand insertion was confirmed by activity detection of octreotide in HSPE-PEG4000-Octreotide with synchronous fluorescence. 1% octreotide density could achieve the best uptake efficiency on NCI-H-446 and SMMC-7721 cell lines among all liposomes. Octreotide-grafted long-circulating liposome (Oct-SSL) was prepared based on 1% octreotide density. The results showed that the insertion of PEG reduced cellular uptake and cytotoxicity. However, Oct-SSL showed a higher MRT compared with Oct-L showing the relative higher long circulating effect in pharmacokinetic study. Oct-SSL also showed tumor growth inhibition of twofold compared with other groups in Heps xenograft model mice. Oct-SSL with suitable ligand density and PEG modification exhibited significant effective tumor targeting for antitumor drug delivery.

Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer.

Given the superior soft tissue contrasts obtained by MRI and the long residence times of magnetic nanoparticles (MNPs) in soft tissues, MNP-based theranostic systems are being developed for simultaneous imaging and treatment. However, development of such theranostic nanoformulations presents significant challenges of balancing the therapeutic and diagnostic functionalities in order to achieve optimum effect from both. Here we developed a simple theranostic nanoformulation based on magnetic nanoclusters (MNCs) stabilized by a bisphosphonate-modified poly(glutamic acid)-b-(ethylene glycol) block copolymer and complexed with cisplatin. The MNCs were decorated with luteinizing hormone releasing hormone (LHRH) to target LHRH receptors (LHRHr) overexpressed in ovarian cancer cells. The targeted MNCs significantly improved the uptake of the drug in cancer cells and decreased its IC50 compared to the nontargeted formulations. Also, the enhanced LHRHr-mediated uptake of the targeted MNCs resulted in enhancement in the T2-weighted negative contrast in cellular phantom gels. Taken together, the LHRH-conjugated MNCs show good potential as ovarian cancer theranostics.

Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity.

Modification of hydrophilic proteins with amphiphilic block copolymers capable of crossing cell membranes is a new strategy to improve protein delivery to the brain. Leptin, a candidate for the treatment of epidemic obesity, has failed in part because of impairment in its transport across the blood-brain barrier (BBB) that develops with obesity. We posit that modification of leptin with poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), Pluronic P85 (P85) might permit this protein to penetrate the BBB independently of its transporter, thereby overcoming peripheral leptin resistance. Here we report that peripherally administered leptin-P85 conjugates exhibit biological activity by reducing food intake in mouse models of obesity (ob/ob, and diet-induced obese mouse). We further generated two new leptin-P85 conjugates: one, Lep(ss)-P85(L), containing one P85 chain and another, Lep(ss)-P85(H), containing multiple P85 chains. We report data on their purification, analytical characterization, peripheral and brain pharmacokinetics (PK). Lep(ss)-P85(L) crosses the BBB using the leptin transporter, and exhibits improved peripheral PK along with increased accumulation in the brain compared to unmodified leptin. Lep(ss)-P85(H) also has improved peripheral PK but in a striking difference to the first conjugate penetrates the BBB independently of the leptin transporter via a non-saturable mechanism. The results demonstrate that leptin analogs can be developed through chemical modification of the native leptin with P85 to overcome leptin resistance at the level of the BBB, thus improving the potential for the treatment of obesity.

Effect of octreotide-polyethylene glycol(100) monostearate modification on the pharmacokinetics and cellular uptake of nanostructured lipid carrier loaded with hydroxycamptothecine.

A new conjugate, octreotide-polyethylene glycol(100) monostearate (OPMS), was developed for the enhancement of targeting delivery of hydroxycamptothecine (HCPT) loaded in nanostructured lipid carrier (NLC). 2 × 10(-3) and 5 × 10(-3) mmol of OPMS were respectively used to modify NLC so that the targeted nanocarriers with low and high ligand density were obtained. For comparison, the pegylated NLCs without octreotide were prepared by adding equal molar amounts of polyethylene glycol(100) monostearate (PGMS). The relation between the modification levels and properties of various NLCs were studied in vivo and in vitro. At a high modification level, a slower release rate of HCPT and the more stable nanocarriers was achieved. At the same time, the fixed aqueous layer thickness (FALT) and average surface density of PEG chains (SD(PEG)) was increased, but the distance (D) between two neighboring PEG grafting sites became narrower. The in vivo pharmacokinetic study in healthy rat indicated that the modified NLCs had a longer circulation than NLC (P < 0.05) due to pegylation effect and OPMS modified NLCs had larger MRT and AUC(0-t) than that of PGMS modified NLCs at the same modification level. Furthermore, the florescence microscopy observation also showed the targeting effect of octreotide modification on somatostatin receptors (SSTRs) of tumor cell (SMMC-7721). The uptake of SMMC-7721 was much more than that of normal liver cell (L02) for OPMS modified NLC, and the highest uptake was observed for 5 × 10(-3) mmol of OPMS modified one. No obvious difference was found among the L02 uptake of OPMS modified NLCs and NLC, but their uptake was higher than that of PGMS modified NLCs. All the results indicated that the OPMS highly modified NLCs would improve the effect of antitumor therapy by inhibiting the degradation, evading RES and enhancing the drug uptake of tumor cells.

A novel lipoprotein-mimic nanocarrier composed of the modified protein and lipid for tumor cell targeting delivery.

Ursodeoxycholic acid (UA) modified protein-lipid nanocomplex (uP-LNC) as a novel biomimetic nanocarrier was developed for tumor-targeting delivery. Bovine serum albumin (BSA) was used as a model protein and its amino groups were covalently modified by UA. Lipid nanoparticle (LNP) composed of phospholipids, triglycerides and octadecylamine was prepared by using solvent evaporation method and was used as the core. UA modified BSA (uP) was attached onto the surface of LNP by post-insert method and generated the protein-lipid nanocomplex. As the control, cholesteryl hemiglutarate (CH), a non-targeting ligand was also used to modify BSA and then formed CH modified protein-lipid nanocomplex (cP-LNC). The combining efficiency of modified BSA with LNP, determined by Bradford protein assay, increased with the enhancement of substitution degree. The modified BSA and nanocomplex were characterized for the substitute degree, average molecular weight, surface tension, particle size and zeta potential by various physicochemical analyses. In vitro dissolution tests and cell uptake studies were performed by loading coumarin-6 as a fluorescent probe. The results indicated that the UA modified protein attached on the nanoparticles significantly decreased drug release from the nanocomplex in pH 7.4 medium, The uptake of uP-LNC was higher in hepatic carcinoma cells (HepG2 and Bel 7402) than in normal liver cells (L02). Furthermore, the uptake of uP-LNC was significantly higher than that of cP-LNC and LNP in these cells. The uptake was dependent on time, temperature and concentration, and could be inhibited by free UA. In addition, the MTT assay of uP-LNC and u(x)P with various degrees of substitution showed very low cytotoxicity at tested concentrations in all cells. The UA modification served to facilitate the specific receptor and energy mediated endocytosis process of the protein-lipid nanocomplex and enabled this nanocomplex to be a potential nanocarrier for tumor-targeting drug delivery.