Peritoneal B Cells Play a Role in the Production of Anti-polyethylene Glycol (PEG) IgM against Intravenously Injected siRNA-PEGylated Liposome Complexes.

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

Impact of Anti-PEG IgM Induced via the Topical Application of a Cosmetic Product Containing PEG Derivatives on the Antitumor Effects of PEGylated Liposomal Antitumor Drug Formulations in Mice.

Poly(ethylene glycol) (PEG) is used in many common products, such as cosmetics. PEG, however, is also used to covalently conjugate drug molecules, proteins, or nanocarriers, which is termed PEGylation, to serve as a shield against the natural immune system of the human body. Repeated administration of some PEGylated products, however, is known to induce anti-PEG antibodies. In addition, preexisting anti-PEG antibodies are now being detected in healthy individuals who have never received PEGylated therapeutics. Both treatment-induced and preexisting anti-PEG antibodies alter the pharmacokinetic properties, which can result in a subsequent reduction in the therapeutic efficacy of administered PEGylated therapeutics through the so-called accelerated blood clearance (ABC) phenomenon. Moreover, these anti-PEG antibodies are widely reported to be related to severe hypersensitivity reactions following the administration of PEGylated therapeutics, including COVID-19 vaccines. We recently reported that the topical application of a cosmetic product containing PEG derivatives induced anti-PEG immunoglobulin M (IgM) in a mouse model. Our finding indicates that the PEG derivatives in cosmetic products could be a major cause of the preexistence of anti-PEG antibodies in healthy individuals. In this study, therefore, the pharmacokinetics and therapeutic effects of Doxil (doxorubicin hydrochloride-loaded PEGylated liposomes) and oxaliplatin-loaded PEGylated liposomes (Liposomal l-OHP) were studied in mice. The anti-PEG IgM antibodies induced by the topical application of cosmetic products obviously accelerated the blood clearance of both PEGylated liposomal formulations. Moreover, in C26 tumor-bearing mice, the tumor growth suppressive effects of both Doxil and Liposomal l-OHP were significantly attenuated in the presence of anti-PEG IgM antibodies induced by the topical application of cosmetic products. These results confirm that the topical application of a cosmetic product containing PEG derivatives could produce preexisting anti-PEG antibodies that then affect the therapeutic efficacy of subsequent doses of PEGylated therapeutics.

Acute Kidney Injury Caused by Rhabdomyolysis Is Ameliorated by Serum Albumin-Based Supersulfide Donors through Antioxidative Pathways.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

A polyethylene glycol-conjugate of deoxycytidine analog, DFP-14927, produces potential antitumor effects on pancreatic tumor-xenograft murine models via inducing G2/M arrest.

A deoxycytidine analog is a potential agent for the treatment of several cancers, which includes poorly prognostic pancreatic cancer. We previously developed deoxycytidine analog DFP-10917, and long-term/low-dose infusions of this analog has produced antitumor effects in leukemia cancer- and ovarian cancer-xenograft models. DFP-10917 is now undergoing clinical Phase III study in the United States for the treatment of patients with relapsed or refractory acute myeloid leukemia. PEG-drug conjugation has become a promising technique to improve the pharmacokinetic and pharmacodynamic properties of anti-cancer drugs. In the present study, we synthesized a novel PEG-drug conjugate of DFP-10917, referred to hereafter as DFP-14927, using a 4-armed CTPEG system to endow the DFP-10917 drug with favorable long-circulating properties that maximize its utility and antitumor efficacy. Intravenous injection of the synthesized DFP-14927 returned encouraging antitumor effects in a Panc-1 human pancreatic tumor- and a BxPC-3 human pancreatic tumor-xenograft models. These effects were comparable to that of free DFP-10917 as well as to that of gemcitabine, which is considered a standard in the treatment of pancreatic cancer. In vitro studies revealed that DFP-14927 inhibits cell division on human pancreatic cancer cell lines via arrest of the G2/M phase in the cell cycle, which is consistent with the effects of free DFP-10917. Intravenous administration of the newly synthesized DFP-14927 has induced G2/M arrest in human pancreatic tumor-xenograft murine models, which represents an improvement in the pharmacokinetics of DFP-10917. DFP-14927 could be an alternative for patients who cannot accept prolonged or continuous infusions of DFP-10917.

Ionic Liquid-Based Transcutaneous Peptide Antitumor Vaccine: Therapeutic Effect in a Mouse Tumor Model.

Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.

Wnt2b and Wnt5a expression is highly associated with M2 TAMs in non­small cell lung cancer.

Tumor­associated macrophages (TAMs), particularly M2 macrophages, promote tumor progression, while Wnt genes encode a family of multi­functional glycoproteins that serve an important role in tumorigenesis. Immunohistochemical studies were performed to evaluate Wnt2b and Wnt5a expression in tumor and stromal cells in M2 and M1 TAMs and Ki­67 proliferation index in 160 consecutive patients with resected non­small cell lung cancer (NSCLC). Overall, 52 tumors (32.5%) were classified as tumoral Wnt2b­high (Wnt2b­positive tumor cells >30%) and 95 (59.4%) as stromal Wnt2b­high (Wnt2b­positive stromal cells >30%), while 75 (46.9%) were classified as tumoral Wnt5a­high (Wnt5a­positive tumor cells >30%) and 63 (39.4%) as stromal Wnt5a­high (Wnt5a­positive stromal cells >28%). The density of M2 TAMs was significantly higher in the tumoral (P=0.0024) and stromal Wnt2b­high groups (P=0.0054). The density of M2 TAMs was also significantly higher in the tumoral (P=0.0005) and stromal Wnt5a­high groups (P=0.0486). By contrast, no difference in stromal or islet M1 TAM density was observed in relation to tumoral or stromal Wnt2b or Wnt5a status. Furthermore, Ki­67 proliferation index was significantly higher in the tumoral (P=0.0121) and stromal Wnt2b­high (P=0.0019) and tumoral Wnt5a­high (P=0.0088) groups. Overall survival rate was significantly lower in the Wnt2b­high (P=0.0437), Wnt5a­high (P=0.0106) and M2 TAM­high (P=0.0060) groups. Wnt2b and Wnt5a expression in tumor and stromal cells may induce M2 TAMs to produce more aggressive behavior during tumor progression in NSCLC.

Oral administration of sodium bicarbonate can enhance the therapeutic outcome of Doxil® via neutralizing the acidic tumor microenvironment.

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 (NaHCO3) can neutralize the acidic tumor microenvironment and enhance the effects of small molecule anticancer drugs. However, it is not known whether or not increasing the tumor pH by oral administration of NaHCO3 leads to enhanced antitumor effects of lipidic nanoparticle formulations of weak-base anticancer drugs, such as Doxil®. In this study, we investigated the antitumor efficacy of Doxil® in combination with oral administration of NaHCO3 in a Colon26 tumor-bearing mouse model. NaHCO3 clearly enhanced the tumor-growth inhibitory effect of Doxil® without exacerbating any systemic side effects. In vitro studies indicated that high levels of DXR were internalized into cells at neutral pH. These studies demonstrate that the neutralization of acidic tumor microenvironment by an oral administration of NaHCO3 could be a promising approach to enhance the therapeutic outcomes of Doxil®.

Development of an Antigen Delivery System for a B Cell-Targeted Vaccine as an Alternative to Dendritic Cell-Targeted Vaccines.

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.

The Challenge to Deliver Oxaliplatin (l-OHP) to Solid Tumors: Development of Liposomal l-OHP Formulations.

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.

A mouse model for studying the effect of blood anti-PEG IgMs levels on the in vivo fate of PEGylated liposomes.

The presence of anti-polyethylene glycol (PEG) antibodies in the systemic circulation might have potential implications for the therapeutic activity of PEGylated products in vivo in the clinic. In order to study the effect of pre-existing anti-PEG antibodies on the in vivo fate and the therapeutic efficiency of PEGylated therapeutics, we developed a BALB/c mouse model by virtue of the intraperitoneal (i.p.) inoculation of hybridoma cells (HIK-M09 and HIK-M11), secreting monoclonal anti-PEG IgM, mimicking the presence of pre-existing anti-PEG antibodies in the blood. In the model, the titers of anti-PEG IgM in the blood increased as a function of hybridoma cells numbers and time after i.p. inoculation. The in vivo levels of anti-PEG IgM decreased in a dose-dependent manner, following i.v. administration of empty PEGylated liposomes. C26 tumor-bearing mice with measurable levels of anti-PEG IgM, receiving i.v. injection of DiR-labeled empty PEGylated liposomes, showed lower levels of liposomal tumor accumulation and higher levels of liver and spleen accumulation, compared to C26 tumor-bearing mice without measurable anti-PEG IgM. This specifies that the presence of anti-PEG IgM in the murine circulation induced accelerated blood clearance of PEGylated liposomes and reduced their tumor accumulation. The biodistribution and antitumor efficacy of commercially available doxorubicin (DXR)-containing PEGylated liposomes, Doxil®, were scrutinized in the anti-PEG IgM mouse model. In C26 tumor-bearing mice having circulating anti-PEG IgM, at 24 h after injection almost no DXR was observed in blood and tumor, and increased DXR accumulation was observed in spleen and liver, compared to tumor-bearing mice with no circulating anti-PEG IgM. The antitumor efficacy of Doxil® was significantly compromised in the C26 tumor-bearing mice in the presence of anti-PEG IgM. These results demonstrate that the anti-PEG IgM mouse model could be a useful prognostic indicator for the therapeutic effectiveness of different formulations of PEGylated therapeutics in pre-clinical studies.

A novel polyethylene glycol (PEG)-drug conjugate of Venetoclax, a Bcl-2 inhibitor, for treatment of acute myeloid leukemia (AML).

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.

I.p.-injected cationic liposomes are retained and accumulate in peritoneally disseminated tumors.

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.

The Therapeutic Effect of Human Serum Albumin Dimer-Doxorubicin Complex against Human Pancreatic Tumors.

Human serum albumin (HSA) is a versatile drug carrier with active tumor targeting capacity for an antitumor drug delivery system. Nanoparticle albumin-bound (nab)-technology, such as nab-paclitaxel (Abraxane®), has attracted significant interest in drug delivery research. Recently, we demonstrated that HSA dimer (HSA-d) possesses a higher tumor distribution than HSA monomer (HSA-m). Therefore, HSA-d is more suitable as a drug carrier for antitumor therapy and can improve nab technology. This study investigated the efficacy of HSA-d-doxorubicin (HSA-d-DOX) as next-generation nab technology for tumor treatment. DOX conjugated to HSA-d via a tunable pH-sensitive linker for the controlled release of DOX. Lyophilization did not affect the particle size of HSA-d-DOX or the release of DOX. HSA-d-DOX showed significantly higher cytotoxicity than HSA-m-DOX in vitro. In the SUIzo Tumor-2 (SUIT2) human pancreatic tumor subcutaneous inoculation model, HSA-d-DOX could significantly inhibit tumor growth without causing serious side effects, as compared to the HSA binding DOX prodrug, which utilized endogenous HSA as a nano-drug delivery system (DDS) carrier. These results indicate that HSA-d could function as a natural solubilizer of insoluble drugs and an active targeting carrier in intractable tumors with low vascular permeability, such as pancreatic tumors. In conclusion, HSA-d can be an effective drug carrier for the antitumor drug delivery system against human pancreatic tumors.

Liposomalization of Oxaliplatin Exacerbates the Non-Liposomal Formulation-Induced Decrease of Sweet Taste Sensitivity in Rats.

Here, we investigated whether or not the characteristics of the oxaliplatin-induced sweet taste sensitivity were altered by PEGylated liposomalization of oxaliplatin (liposomal oxaliplatin), which enhances its anticancer efficacy. Liposomal oxaliplatin and oxaliplatin were intravenously and intraperitoneally, respectively, administered to male Sprague-Dawley rats at the total dose of 8 mg/kg. A brief-access test for evaluation of sweet taste sensitivity on day 7 revealed that both liposomal oxaliplatin and oxaliplatin decreased the sensitivity of rats, the degree with the former being greater than in the case of the latter. Liposomalization of oxaliplatin increased the accumulation of platinum in lingual non-epithelial tissues, through which taste nerves passed. The lingual platinum accumulation induced by not only liposomal oxaliplatin but also oxaliplatin was decreased on cooling of the tongue during the administration. In the current study, we revealed that liposomalization of oxaliplatin exacerbated the oxaliplatin-induced decrease of sweet taste sensitivity by increasing the accumulation of platinum/oxaliplatin in lingual non-epithelial tissues. These findings may suggest that reduction of liposomal oxaliplatin distribution to the tongue on cooling during the administration prevents exacerbation of the decrease of sweet taste sensitivity, maintaining the quality of life and chemotherapeutic outcome in patients.

Doxorubicin Embedded into Nanofibrillated Bacterial Cellulose (NFBC) Produces a Promising Therapeutic Outcome for Peritoneally Metastatic Gastric Cancer in Mice Models via Intraperitoneal Direct Injection.

Natural materials such as bacterial cellulose are gaining interest for their use as drug-delivery vehicles. Herein, the utility of nanofibrillated bacterial cellulose (NFBC), which is produced by culturing a cellulose-producing bacterium (Gluconacetobacter intermedius NEDO-01) in a medium supplemented with carboxymethylcellulose (CMC) that is referred to as CM-NFBC, is described. Recently, we demonstrated that intraperitoneal administration of paclitaxel (PTX)-containing CM-NFBC efficiently suppressed tumor growth in a peritoneally disseminated cancer xenograft model. In this study, to confirm the applicability of NFBC in cancer therapy, a chemotherapeutic agent, doxorubicin (DXR), embedded into CM-NFBC, was examined for its efficiency to treat a peritoneally disseminated gastric cancer via intraperitoneal administration. DXR was efficiently embedded into CM-NFBC (DXR/CM-NFBC). In an in vitro release experiment, 79.5% of DXR was released linearly into the peritoneal wash fluid over a period of 24 h. In the peritoneally disseminated gastric cancer xenograft model, intraperitoneal administration of DXR/CM-NFBC induced superior tumor growth inhibition (TGI = 85.5%) by day 35 post-tumor inoculation, compared to free DXR (TGI = 62.4%). In addition, compared with free DXR, the severe side effects that cause body weight loss were lessened via treatment with DXR/CM-NFBC. These results support the feasibility of CM-NFBC as a drug-delivery vehicle for various anticancer agents. This approach may lead to improved therapeutic outcomes for the treatment of intraperitoneally disseminated cancers.

Increasing Tumor Extracellular pH by an Oral Alkalinizing Agent Improves Antitumor Responses of Anti-PD-1 Antibody: Implication of Relationships between Serum Bicarbonate Concentrations, Urinary pH, and Therapeutic Outcomes.

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.

Anti-PEG IgM production and accelerated blood clearance phenomenon after the administration of PEGylated exosomes in mice.

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.

Evidence for Delivery of Abraxane via a Denatured-Albumin Transport System.

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.

Therapeutic efficacy of a paclitaxel-loaded nanofibrillated bacterial cellulose (PTX/NFBC) formulation in a peritoneally disseminated gastric cancer xenograft model.

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.

Neutralization of Acidic Tumor Microenvironment (TME) with Daily Oral Dosing of Sodium Potassium Citrate (K/Na Citrate) Increases Therapeutic Effect of Anti-cancer Agent in Pancreatic Cancer Xenograft Mice Model.

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.