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Vaccines have contributed to the prevention of infectious diseases for a long time. Pathogen-derived antigens and adjuvants in vaccine formulations stimulate immune cells to elicit humoral and cellular immune responses against pathogens. Achieving highly immune responses with decreased adverse effects requires the development of a system that can deliver antigens to specific immune cells. Dendritic cells (DCs) are well-known professional antigen presenting cells (APCs) that initiate acquired immune responses by presenting antigens to T cells. Accordingly, DC-targeted vaccines have been investigated and applied in clinical trials for the treatment of infectious diseases and for chronic diseases such as cancers. In addition to DCs, B lymphocytes are regarded as professional APCs despite their primary role in humoral immunity. Therefore, B cell-targeted vaccines are also expected to elicit both humoral and cellular immune responses. In this review we summarize the basic functions of DCs and B cells as APCs. We also provide information on DC and B cell targeted vaccines in preclinical and clinical settings. Finally, we introduce our novel antigen delivery system that targets splenic marginal zone B cells and the ability of this system to act as a novel vaccine that elicits both humoral and cellular immune responses.

Intraperitoneal (i.p) chemotherapy is an attractive approach to treat peritoneally disseminated cancers by delivering therapeutic agents directly to the peritoneal cavity where some disseminated tumors are located. Cationic liposomes (CLs) have been used as a viable delivery carrier for i.p. chemotherapy to improve the peritoneal retention of anticancer agents. However, there are no reports on the fate of CLs following i.p. administration to the peritoneal cavity in the presence of disseminated tumors. We prepared a tumor xenograft murine model of peritoneally disseminated gastric cancer by i.p. inoculation of human gastric cancer cells and followed the fate of either CLs or PEGylated CLs (PEG-CLs) after i.p. injection in the model. I.p.-injected CLs were retained in peritoneal cavity for at least 3 days post-injection as a result of clustering with ascites fluid proteins, mainly albumin, while i.p. PEG-CLs was rapidly cleared from the peritoneal cavity to the circulation within 3 h post-injection. Importantly, i.p. CLs efficiently accumulated in the targeted disseminated tumor cells, but not in other abdominal organs including liver, spleen, and kidney. The tumor selectivity upon i.p. administration of CLs may be associated with the lymphatic drainage system. A lipoplex formulation composed of CLs with short hairpin RNA (shRNA) against luciferase, a model therapeutic agent, suppressed luciferase activity in peritoneally disseminated tumors by 80%, with no cytokine secretion in serum. This suggests that i.p. CLs can efficiently deliver a therapeutic agent to peritoneally disseminated tumors with few systemic adverse events. These results suggest that i.p. treatment with CLs or non-PEGylated lipoplexes may be a promising approach for the treatment of peritoneally disseminated cancers through their ability to selectively deliver therapeutic agents to i.p. target sites with minimal systemic adverse events.

PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH

Tumor‑associated macrophages (TAMs), particularly M2 macrophages, promote tumor progression, while

Palau'amine has received a great deal of attention as an attractive synthetic target due to its intriguing molecular architecture and significant immunosuppressive activity, and we achieved its total synthesis in 2015. However, the synthesized palau'amine has not been readily applicable to the mechanistic study of immunosuppressive activity, because it requires 45 longest linear steps from a commercially available compound. Here, we report the short-step construction of the ABCDEF hexacyclic ring core of palau'amine. The construction of the CDE tricyclic ring core in a single step is achieved by our pKa concept for proceeding with unfavorable equilibrium reactions, and a palau'amine analog without the aminomethyl and chloride groups is synthesized in 20 longest linear steps from the same starting material. The palau'amine analog is confirmed to retain the immunosuppressive activity. The present synthetic approach for a palau'amine analog has the potential for use in the development of palau'amine probes for mechanistic elucidation.

Acidic extracellular pH (pHe) is characteristic of the tumor microenvironment. Several reports suggest that increasing pHe improves the response of immune checkpoint inhibitors in murine models. To increase pHe, either sodium bicarbonate (NaHCO3) or citric acid/potassium-sodium citrate (KNa-cit) was chronically administered to mice. It is hypothesized that bicarbonate ions (HCO3-), produced from these alkalinizing agents in vivo, increased pHe in the tumor, and excess HCO3- eliminated into urine increased urinary pH values. However, there is little published information on the effect of changing serum HCO3- concentrations, urinary HCO3- concentrations and urinary pH values on the therapeutic outcomes of immunotherapy. In this study, we report that oral administration of either NaHCO3 or KNa-cit increased responses to anti-programmed cell death-1 (PD-1) antibody, an immune checkpoint inhibitor, in a murine B16 melanoma model. In addition, we report that daily oral administration of an alkalinizing agent increased blood HCO3- concentrations, corresponding to increasing the tumor pHe. Serum HCO3- concentrations also correlated with urinary HCO3- concentrations and urinary pH values. There was a clear relationship between urinary pH values and the antitumor effects of immunotherapy with anti-PD-1 antibody. Our results imply that blood HCO3- concentrations, corresponding to tumor pHe and urinary pH values, may be important factors that predict the clinical outcomes of an immunotherapeutic agent, when combined with alkalinizing agents such as NaHCO3 and KNa-cit.

Recently, there is an increasing interest in exosomes or extracellular vesicles as potential candidates for delivering RNAs, proteins, genes, and anticancer agents. Engineering of exosome properties is rapidly evolving as a means of expanding exosome applications. PEGylation of exosomes is a technique used to improve their in vivo stability, circulation half-lives, and sometimes to allow the binding targeting ligands to the exosome exterior. According to FDA guidelines for the development of PEGylated proteins, immunological responses to PEGylated molecules and particles should be examined. In this study, we prepared PEGylated exosomes and investigated the production of anti-PEG IgM antibodies after single i.v. injections in mice. In addition, we monitored blood concentrations and tumor accumulation of a second dose of PEGylated exosomes administered after the initial dose. Single injections of PEGylated exosomes in mice induced anti-PEG IgM production in a T cell-dependent manner. The anti-PEG IgM production decreased when the injection dose of PEGylated exosomes was further increased. Anti-PEG IgM induced by injection of PEGylated exosomes decreased blood concentrations of a second dose of PEGylated exosomes and suppressed their tumor accumulation in a C26 murine colorectal cancer model. Initial injection doses of either PEGylated liposomes or PEGylated ovalbumin (PEG-OVA), both of them induced anti-PEG IgM production, also decreased the blood concentration of PEGylated exosomes. Interestingly, anti-PEG IgM induced by injection of PEGylated exosomes did not affect the blood concentration of PEG-OVA. These results imply the importance of monitoring anti-PEG IgM when repeat PEGylated exosome doses are required and/or when PEGylated exosomes are used together with other PEGylated therapeutics.

Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.

Polyethylene glycol (PEG) is a versatile polymer that is widely used as an additive in foods and cosmetics, and as a carrier in PEGylated therapeutics. Even though PEG is thought to be less immunogenic, or perhaps even non-immunogenic, with a variety of physicochemical properties, there is mounting evidence that PEG causes immunogenic responses when conjugated with other materials such as proteins and nanocarriers. Under these conditions, PEG with other materials can result in the production of anti-PEG antibodies after administration. The antibodies that are induced seem to have a deleterious impact on the therapeutic efficacy of subsequently administered PEGylated formulations. In addition, hypersensitivity to PEGylated formulations could be a significant barrier to the utility of PEGylated products. Several reports have linked the presence of anti-PEG antibodies to incidences of complement activation-related pseudoallergy (CARPA) following the administration of PEGylated formulations. The use of COVID-19 mRNA vaccines, which are composed mainly of PEGylated lipid nanoparticles (LNPs), has recently gained wide acceptance, although many cases of post-vaccination hypersensitivity have been documented. Therefore, our review focuses not only on the importance of PEGs and its great role in improving the therapeutic efficacy of various medications, but also on the hypersensitivity reactions attributed to the use of PEGylated products that include PEG-based mRNA COVID-19 vaccines.

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

Pegfilgrastim is a recombinant PEGylated human granulocyte colony-stimulating factor (G-CSF) analog filgrastim (trade names Neulasta® or G-Lasta®) that stimulates the production of white blood cells (neutrophils). It is employed as an alternative to filgrastim (G-CSF) for chemotherapy-induced neutropenia in patients due to its longer half-life. In clinical settings, PEG-G-CSF is administered to cancer patients via both the s.c. and i.v. routes. In a murine study, we showed that, regardless of administration route, initial doses of PEG-G-CSF above 0.06 mg/kg elicited anti-PEG immune response in a dose-dependent manner. I.v. administration elicited higher levels of anti-PEG IgM than the s.c. route. Initial doses of PEG-G-CSF (6 mg/kg) that were high enough to trigger production of anti-PEG IgM, did not trigger the accelerated clearance of a lower subsequent dose (0.06 mg/kg) that was similar to i.v. clinical doses of PEG-G-CSF, but when the subsequent dose of PEG-G-CSF was raised to (6 mg/kg), the initial dose triggered the accelerated clearance of the second dose via an anti-PEG IgM-mediated complement activation. Similar observations were noted when an increased PEG-OVA dose was given as the second dose, indicating that pre-existing and/or treatment-induced anti-PEG antibodies might compromise the therapeutic activity and/or reduce tolerance of other PEGylated formulations. To the best of our knowledge, this is the first report to suggest the induction of the ABC phenomenon upon repeated injections of pegfilgrastim. In the clinic, cancer patients, receiving multiple cycles of chemotherapy, receive multiple cycles of pegfilgrastim to avoid infections and substantial morbidity. The ABC phenomenon to pegfilgrastim appears to be the cause of loss of clinical benefit of sequential treatments with pegfilgrastim in patients.

PEGylation had been used successfully to improve the circulation half-lives and some physicochemical properties of protein therapeutics. However, anti-polyethylene glycol (anti-PEG) antibodies, either pre-existing or treatment-induced, can negatively affect the pharmacokinetics and pharmacological efficacy of PEGylated proteins. We have examined anti-PEG immune responses in mice for peginterferon alfa-2a (Pegasys), a clinically approved PEGylated protein therapeutic, at both the recommended dose (equivalent to 3 μg/kg in mice) and at higher doses (150 μg/kg) for single or repeated subcutaneous (s.c.) administrations. The effect of treatment-induced anti-PEG IgM on serum concentrations of Pegasys, following repeated administrations, was evaluated. In addition, the effect of pre-existing anti-PEG IgM elicited by a different PEGylated protein, PEG-OVA, on the systemic clearance of Pegasys, was investigated. At a s.c. dose of 3 μg/kg, single injections of Pegasys barely elicited anti-PEG immune responses. Four repeated doses of 150 μg/kg Pegasys elicited anti-PEG IgM production, depending on dose frequency, and triggered the rapid clearance of subsequent doses. In addition, anti-PEG-IgM produced in response to prior administration of PEG-OVA caused a rapid blood clearance of Pegasys. Our results, therefore, underscore the importance of screening for both pre-existing and treatment-induced anti-PEG antibodies in patients prior to and during treatment with PEGylated protein drugs.

Oxaliplatin (l-OHP) is a third-generation platinum (Pt) agent approved for the treatment of patients with advanced colorectal cancer. Despite the fact that l-OHP has shown clinical therapeutic efficacy and better tolerability compared with other Pt agents, the use of l-OHP has been limited to clinical settings because of dose-limiting side effects such as cumulative neurotoxicity and acute dysesthesias, which can be severe. In preclinical and clinical studies, our group and several others have attempted the delivery of l-OHP to solid tumors via encapsulation in PEGylated liposomes. Herein, we review these attempts.

The pH of the tumor microenvironment in solid tumors is reported to be more acidic than that of normal tissues. The pH is controlled by over-expression of several transporters that are associated with the progression, angiogenesis, and metastasis of solid tumors. Antitumor effects of weak-base anticancer agents, such as doxorubicin (DXR), could be reduced in an acidic environment because of increases in the ionized form of the drug under these conditions, reducing its membrane penetrability. In our previous studies, we demonstrated that oral administration of sodium bicarbonate (NaHCO

Many macromolecular antitumor drugs were developed based on the enhanced permeability and retention (EPR) effect, for example, albumin-bound paclitaxel nanoparticles (nab-PTX and Abraxane) and pegylated liposomal doxorubicin (Doxil). However, these EPR effect-based therapeutic systems are less effective in malignant tumors with low vascular permeability, such as pancreatic tumors. Because the EPR effect depends on nanoparticles' size, we first determined nanoparticles' size associated with a high tumor-targeting rate in a human pancreatic tumor xenograft model with low vascular permeability. Abraxane appears to behave as an albumin monomer (7 nm) in the blood circulation following intravenous injection. The

The purpose of this study was to develop a Bio-layer interferometry (BLI) system that could be an alternative approach for the direct evaluation of anti-polyethylene glycol (PEG) immunoglobulin M (IgM)-mediated complement activation of the accelerated blood clearance (ABC) phenomenon. Complement activation is well known to play an important role in the clearance of PEGylated and non-PEGylated nanomedicines following intravenous injection. This complement system is also thought to be responsible for the ABC phenomenon wherein repeated injections of PEGylated products are bound by anti-PEG antibodies. This study used three different sources of anti-PEG antibodies: HIK-M09 monoclonal antibodies (mAbs); HIK-M11 mAbs; and antiserum containing polyclonal anti-PEG IgMs. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG

In the development of drug delivery system (DDS)-based anticancer drugs, the techniques for the intratumor mapping and quantification of active pharmaceutical ingredients (API) in pharmaceuticals must be pivotal for predicting pharmacological effects and adverse events. X-ray fluorescence spectrometry (XRF) is a potent analytical tool for mapping/quantifying platinum pharmaceutics such as oxaliplatin (l-OHP) and its liposomal formulation. In recent studies, we employed XRF to visualize the intratumor micro-distribution of l-OHP in a tumor-bearing model mouse intravenously injected with either free l-OHP or l-OHP liposomes. The intratumor distribution of l-OHP within tumor sections could be determined by XRF to detect platinum atoms. After treatment with the liposomal formulation, the l-OHP was localized near the tumor vessels and, via repeated injections, increasingly accumulated in tumors by a much greater degree than treatment with free l-OHP. The repeated injections of l-OHP liposomes improved the vascular permeability via inducing the apoptosis of tumor cells near the tumor vessels, which should improve the tumor microenvironment and enhance the intratumor accumulation of repeated doses of l-OHP liposomes. The proposed process was also used to visualize the intratumor distribution of l-OHP in rectal cancer specimens resected from a patient who had received l-OHP-based preoperative chemotherapy. We further revealed that neutralization of an acidic tumor microenvironment via oral administration with NaHCO3 could improve the therapeutic efficacy of weakly basic anticancer agent-encapsulating liposomes. Collectively, mapping/quantifying the intratumor API in DDS drugs and/or improving the tumor microenvironment would be an effective means to accelerate the clinical development of DDS-based anticancer drugs.

RNA interference (RNAi), a potent post-transcriptional gene-silencing action, has received considerable attentions as a novel therapeutic tool to treat intractable cancers. In recent days, we have developed a novel RNAi-based therapeutic formulation, DFP-10825, for the treatment of intractable advanced cancers developed in coelomic cavities. DFP-10825 was composed of chemically synthesized short hairpin RNA (shRNA) against thymidylate synthase (TS), a key enzyme for cancer proliferation, and cationic liposomes, and achieved high therapeutic effect on the mouse models of peritoneally disseminated gastric and ovarian cancers and malignant pleural mesothelioma without severe side effects by intracoelomic direct treatment. We further designed a freeze-dried DFP-10825 formulation for mass industrial production. DFP-10825 is undergoing in pre-clinical phase and goes to clinical trials. This review introduces a DFP-10825 formulation, a potent novel RNAi-based therapeutic maximizing the benefit of RNAi molecule (shRNA).

Exosomes are gaining increasing attention as drug delivery vehicles due to their low toxicity and ability to functionally transfer biological cargos between cells. However, the therapeutic applicability of exosomes is partially hampered by a lack of cell-type specificity. In this study, therefore, we investigated the impact of cell-type tropism on the in vivo systemic delivery of exosomes to tumor tissues. Exosomes derived from murine colorectal cancer cells (C26) (C26-Exos) and murine melanoma cells (B16BL6) (B16BL6-Exos) were collected. In vitro cellular uptake of either autologous (C26) or allogeneic (B16BL6) exosomes by C26 tumor cells was determined. In vivo tumor accumulation of each type of exosomes in mice bearing C26 tumors was monitored with an in vivo imaging system (IVIS). In in vitro studies, autologous C26-Exos were more efficiently taken up by C26 cancer cells, compared to allogeneic B16BL6-Exos. For in vivo studies, exosomes were modified with surface polyethylene glycol (PEG) to improve their circulation lifetimes. Although both types of PEGylated exosomes accumulated in C26-tumor tissue, autologous exosomes were preferentially accumulated within C26-tumor tissue compared to allogeneic exosomes. The increased tumor accumulation of autologous PEGylated exosomes was accompanied by the preferential uptake of exosomes by not only C26-tumor cells but also tumor-associated immune cells. This study implies that cancer cell-type tropism is an important factor in the achievement of tumor cell targeting with cancer cell-derived exosomes.

Purpose In advanced gastric cancer, peritoneal dissemination is a life‐threatening mode of metastasis. Since the treatment options with conventional chemotherapy remain limited, any novel therapeutic strategy that could control such metastasis would improve the outcome of treatment. We recently developed a unique RNA interference therapeutic regimen (DFP‐10825) consisting of short hairpin RNA against thymidylate synthase (TS shRNA) and cationic liposomes. The treatment with DFP‐10825 has shown remarkable antitumor activity in peritoneally disseminated human ovarian cancer–bearing mice via intraperitoneal administration. In this study, we expanded DFP‐10825 to the treatment of peritoneally disseminated gastric cancer. Methods DFP‐10825 was administered intraperitoneally into mice with intraperitoneally implanted human gastric cancer cells (MKN45 or NCI‐N87). Antitumor activity and host survival benefits were monitored. Intraperitoneal distribution of fluorescence‐labeled DFP‐10825 was monitored in this MKN45 peritoneally disseminated mouse model. Results Intraperitoneal injection of DFP‐10825 suppressed tumor growth in two peritoneally disseminated cancer models (MKN45 and NCI‐N87) and increased the survival time of the MKN45 model without severe side effects. Throughout the treatment regimen, no significant body weight loss was associated with the administration of DFP‐10825. Interestingly, after intraperitoneal injection, fluorescence‐labeled DFP‐10825 retained for more than 72 hours in the peritoneal cavity and selectively accumulated in disseminated tumors. Conclusions Intraperitoneal injection of DFP‐10825 demonstrated effective antitumor activity without systemic severe adverse effects via the selective delivery of RNAi molecules into disseminated tumors in the peritoneal cavity. Our current study indicates that DFP‐10825 could become an alternative option to improve the outcomes of patients with peritoneally disseminated gastric cancer., Intraperitoneal DFP‐10825 showed effective antitumor activity in peritoneally disseminated human gastric cancer–bearing mice. As an alternative treatment regimen, DFP‐10825 has the potential to improve the outcomes of patients with peritoneally disseminated gastric cancer.

The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.

RNA interference (RNAi) is one of the most promising strategies for cancer therapeutics. The successful translation of RNAi therapeutics to a clinic setting requires a delivery system that is efficient and simple to upscale. In this study, we devised a simple industrial method to manufacture lipoplex, which includes short hairpin RNA against the expression of thymidylate synthase (TS shRNA) - a key molecule for DNA biosynthesis. An aqueous solution of TS shRNA was gently mixed with either a precursor of cationic liposome (Presome DF-1) or a cationic lipid mixture in an o/w emulsion. This solution was subsequently lyophilized under optimal conditions to obtain either FD-lipoplex-1 or FD-lipoplex-2, respectively. With this method, a lipoplex in activated form was obtained via a simple "one-step" hydration with saline. Both forms of FD-lipoplex showed physicochemical properties comparable to those of conventional lipoplex. FD-lipoplexes stably retained TS shRNA within their formulations in the presence of tumor ascites fluid. Intraperitoneal treatment with either FD-lipoplex-1 or FD-lipoplex-2 provided a therapeutic level of efficacy comparable to that of conventional lipoplex in the treatment of a peritoneal disseminated gastric cancer mouse model. Collectively, established freeze-drying-based methods for RNAi-therapeutic preparation could realistically be used in a clinical setting for the treatment of patients with peritoneal disseminated cancer.

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis.

Palau'amine has received a great deal of attention as an attractive synthetic target due to its intriguing molecular architecture and significant immunosuppressive activity, and we achieved its total synthesis in 2015. However, the synthesized palau'amine has not been readily applicable to the mechanistic study of immunosuppressive activity, because it requires 45 longest linear steps from a commercially available compound. Here, we report the short-step construction of the ABCDEF hexacyclic ring core of palau'amine. The construction of the CDE tricyclic ring core in a single step is achieved by our pKa concept for proceeding with unfavorable equilibrium reactions, and a palau'amine analog without the aminomethyl and chloride groups is synthesized in 20 longest linear steps from the same starting material. The palau'amine analog is confirmed to retain the immunosuppressive activity. The present synthetic approach for a palau'amine analog has the potential for use in the development of palau'amine probes for mechanistic elucidation., A palau'amine analog (2) was synthesized from 2-cyclopentenone in 20 steps. The construction of the CDE tricyclic ring core in a single step is achieved by our pKa concept for proceeding with the unfavorable equilibrium reactions.

Background Venetoclax (VTX) is an anticancer drug. It is a selective Bcl-2 inhibitor that is clinically used for the treatment of patients with lymphomas and leukemias. Treatment with VTX, however, is accompanied by severe adverse events such as tumor lysis syndrome and neutropenia, because VTX readily binds to serum proteins, which results in poor pharmacokinetics and poor tumor tissue concentration. To avoid such adverse events, VTX is administered using a daily or weekly ramp-up schedule that is cumbersome in clinical situations. Aims To overcome these shortcomings, we prepared a novel polyethylene glycol (PEG)-drug conjugate of VTX (PEG-VTX) and evaluated its cytotoxic effects on acute myeloid leukemia (AML) both in vitro and in vivo. Methods and results VTX and 4-armed PEG derivatives were covalently attached through an amide bond linker. In a series of in vitro studies, PEG-VTX selectively induced potent growth inhibition of MV4-11 human AML cells via the inducement of Bcl-2-mediated apoptosis. PEG-VTX had the effect of free VTX, presumably due to the protease-mediated release of VTX from the conjugates. In in vivo studies with AML tumor-xenograft mice models, intravenous PEG-VTX promoted sufficient tumor growth suppression. Compared with a regimen of oral free VTX, the intravenous regimen in those studies used a VTX dosage that was 15-30 times smaller for an OCI-AML-2 xenograft model and a dosing regimen that was less frequent for an MV4-11 xenograft model. The most important development, however, was the absence of weight loss related to severe side effects throughout the treatments. An increase in water solubility and the resultant hydrodynamic size of VTX via PEGylation improved the pharmacokinetics of VTX by avoiding protein interactions and lessening the extravasation from blood. The result was an increase in tumor accumulation and a decrease in the nonspecific distribution of VTX. Conclusion The results of this study suggest that PEG-VTX could be an alternative therapeutic option for the safe and effective treatment of patients with AML.

Abraxane, an albumin-bound paclitaxel nanoparticle formulation, is superior to conventional paclitaxel preparations because it has better efficacy against unresectable pancreatic cancer. Previous reports suggest that this better efficacy of Abraxane than conventional paclitaxel preparation is probably due to its transport through Gp60, an albumin receptor on the surface of vascular endothelial cells. The increased tumor accumulation of Abraxane is also caused by the secreted protein acid and rich in cysteine in the tumor stroma. However, the uptake mechanism of Abraxane remains poorly understood. In this study, we demonstrated that the delivery of Abraxane occurred via different receptor pathways from that of endogenous albumin. Our results showed that the uptake of endogenous albumin was inhibited by a Gp60 pathway inhibitor in the process of endocytosis through endothelial cells or tumor cells. In contrast, the uptake of Abraxane-derived HSA was less affected by the Gp60 pathway inhibitor but significantly reduced by denatured albumin receptor inhibitors. In conclusion, these data indicate that Abraxane-derived HSA was taken up into endothelial cells or tumor cells by a mechanism different from normal endogenous albumin. These new data on distinct cellular transport pathways of denatured albumin via gp family proteins different from those of innate albumin shed light on the mechanisms of tumor delivery and antitumor activity of Abraxane and provide new scientific rationale for the development of a novel albumin drug delivery strategy via a denatured albumin receptor.

Extracellular pH (pHe) of tumor cells is characteristic of tumor microenvironment (TME). Acidic TME impairs the responses of tumors to some anti-cancer chemotherapies. In this study, we showed that daily oral dosing of sodium potassium citrate (K/Na citrate) increased blood HCO3- concentrations, corresponding to increase of HCO3- concentrations and pHs in urine, and neutralized the tumor pHe. Neutralization of acidic TME by alkaline substance like HCO3-, an active metabolite of K/Na citrate, well potentiated the therapeutic effect of anticancer agent TS-1®, an orally active 5-fuluoro-uracil derivative, in Panc-1 pancreatic cancer-xenograft murine model. Neutralization of acidic TME by using an alkaline K/Na citrate is a smart approach for enhancement of the therapeutic effects of anticancer agents for pancreatic cancer in the end stage.

Nano-fibrillated bacterial cellulose (NFBC) is a safe, biocompatible material that can be prepared by culturing a cellulose-producing bacterium in a culture supplemented with carboxymethylcellulose (CMC) or hydroxypropylcellulose (HPC). CM-NFBC and HP-NFBC, prepared using CMC or HPC, show hydrophilicity and amphiphilicity, respectively, and thus they could be useful carriers for hydrophobic anticancer agents such as paclitaxel (PTX). In the present study, we prepared novel PTX formulations for intraperitoneal administration by associating PTX with either CM-NFBC or HP-NFBC and studied their therapeutic efficacy on peritoneally disseminated gastric cancer in a xenograft nude mouse model. Freeze-dried PTX formulations (PTX/CM-NFBC and PTX/HP-NFBC) were quickly reconstituted with saline without any foaming, compared to nanoparticle albumin-bound PTX (nab-PTX, Abraxane®). Both PTX/NFBC formulations extended the mean survival times in our xenograft murine models compared with either free PTX or nab-PTX. The PTX/NFBC formulations reduced systemic side effects of free PTX relating to weight loss. In our disseminated gastric peritoneal cancer model, the PTX/NFBC formulation increased the therapeutic index for PTX by increasing the therapeutic efficacy and decreasing toxicity. NFBCs should receive consideration as improved carriers for the clinical delivery of hydrophobic anticancer drugs such as PTX in malignancies in the abdominal cavity with peritoneal metastasis and dissemination.

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

Splenic marginal zone B (MZ-B) cells have attracted attention as alternative antigen-presenting cells. We recently developed an original delivery system, using PEGylated liposomes (PEG-Lip) to deliver antigens to MZ-B cells. In this system, to induce antigen-specific immunity, empty PEG-Lip and antigen-containing PEG-Lip were intravenously (i.v.) injected sequentially at 3 day intervals. Since complement activation by the second dose is required for the delivery of antigen-containing PEG-Lip to splenic MZ-B cells, we investigated the ability of liposomes, modified with various PEG derivatives having different functional terminal groups (methoxy PEG (CH3O-PEG), hydroxy PEG (HO-PEG) or polyglycerol (PG), to activate the complement system and deliver a model antigen, ovalbumin (OVA), to splenic MZ-B cells in vitro and in vivo. Hydroxy PEG-modified liposomes (HO-PEG-Lip) both activated the complement system in vitro, and facilitated the preferential association of HO-PEG-lip with MZ–B cells in vitro. Manipulating HO-PEG density, in particular a density of 2 mol% in total lipids, significantly enhanced the association of HO-PEG-Lip with splenic MZ-B cells in vivo. Consequently, a single i.v. injection of HO-PEG-Lip (2 mol%) containing OVA induced OVA-specific IgG response. Our immunization system with HO-PEG-Lip, achieved efficient antigen delivery to MZ-B cells after a single i.v. injection, improving on our previous immunization system. This new delivery technique may be an improved, simple, antigen delivery system to MZ-B cells that induces meaningful levels of humoral immune response.

Many small-molecule drugs exhibit poor aqueous solubility, and various approaches have been developed to improve their solubility and delivery. Chemical conjugation of an insoluble drug to a hydrophilic polymer can promote the self-assembly into nanoparticles (NPs) to increase the apparent solubility and improve the pharmacokinetics of the drug. However, majority of the reports in the field disclose only one composition of the conjugate, while accumulating evidence suggests that structure-activity relationship (SAR) studies must be conducted to identify an optimal construct. In this study, we employed a click chemistry platform to robustly conjugate short-chain methoxypolyethylene glycol (mPEG) of three different molecular weights to a small molecule anti-inflammatory drug, gambogic acid (GA), and studied the SAR. NPs formed with mPEG550 and mPEG5000, referred to as NP-550 and NP-5000, respectively, had larger mean diameters (130.0 ± 16.9 nm and 143.0 ± 0.1 nm, respectively) and higher critical micellar concentrations (CMCs, 9.5 μg mL-1 and 10.5 μg mL-1, respectively) compared to NPs formed with mPEG2000 (NP-2000, mean diameter = 97.8 ± 5.0 nm and CMC = 6.6 μg mL-1). NP-2000 and NP-5000 did not cause significant hemolytic toxicity, whereas NP-550 and free GA induced 90% and 60% hemolysis, respectively. NP-2000 was selected for further studies due to its improved safety, small size and low CMC. In cultured inflammatory macrophages, NP-2000 exhibited activity comparable to free GA in suppressing tumor necrosis factor-α. In mice, NP-2000 showed 185-fold improved drug exposure compared to free GA after intraperitoneal delivery. Treatment with free GA showed little anti-inflammatory activity compared to vehicle control in a murine model of rheumatoid arthritis. In contrast, NP-2000 significantly reduced the paw inflammation by 27% from day 15 to day 29. NP-2000 showed no visible signs of toxicity in mice, while free GA elicited significant irritation at the injection site. Our work emphasizes the importance of performing SAR studies for developing an optimal drug-polymer conjugate for self-assembly into NPs. We also demonstrate a unique application of click chemistry to robustly synthesize a small library of conjugates for the SAR study.