Liposomalization of oxaliplatin induces skin accumulation of it, but negligible skin toxicity

Liposomalization causes alteration of the pharmacokinetics of encapsulated drugs, and allows delivery to tumor tissues through passive targeting via an enhanced permeation and retention (EPR) effect. PEGylated liposomal doxorubicin (Doxil(®), Lipo-DXR), a representative liposomal drug, is well-known to reduce cardiotoxicity and increase the anti-tumor activity of DXR, but to induce the hand-foot syndrome (HFS) as a result of skin DXR accumulation, which is one of its severe adverse effects. We have developed a new liposomal preparation of oxaliplatin (l-OHP), an important anti-tumor drug for treatment of colorectal cancer, using PEGylated liposomes (Lipo-l-OHP), and showed that Lipo-l-OHP exhibits increased anti-tumor activity in tumor-bearing mice compared to the original preparation of l-OHP. However, whether Lipo-l-OHP causes HFS-like skin toxicity similar to Lipo-DXR remains to be determined. Administration of Lipo-l-OHP promoted accumulation of platinum in rat hind paws, however, it caused negligible morphological and histological alterations on the plantar surface of the paws. Administration of DiI-labeled empty PEGylated liposomes gave almost the same distribution profile of dyes into the dermis of hind paws with DXR as in the case of Lipo-DXR. Treatment with Lipo-l-OHP, Lipo-DXR, DiI-labeled empty PEGylated liposomes or empty PEGylated liposomes caused migration of CD68(+) macrophages into the dermis of hind paws. These findings suggest that the skin toxicity on administration of liposomalized drugs is reflected in the proinflammatory characteristics of encapsulated drugs, and indicate that Lipo-l-OHP with a higher anti-cancer effect and no HFS may be an outstanding l-OHP preparation leading to an improved quality of life of cancer patients.


Apoptosis induction of Poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition

Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.


Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-Nitrosated Human Serum Albumin Dimer

In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed.


Metronomic S-1 dosing and thymidylate synthase silencing have synergistic antitumor efficacy in a colorectal cancer xenograft model.

Metronomic chemotherapy is currently considered an emerging therapeutic option in clinical oncology. S-1, an oral formulation of Tegafur (TF), a prodrug of 5-fluorouracil (5-FU), is designed to improve the antitumor activity of 5-FU in tandem with reducing its toxicity. Clinically, metronomic S-1 dosing has been approved for the standard first- and second-line treatment of metastatic or advanced stage of colorectal (CRC). However, expression of intratumor thymidylate synthase (TS), a significant gene in cellular proliferation, is associated with poor outcome to 5-FU-based chemotherapeutic regimens. In this study, therefore, we examined the effect of a combination of TS silencing by an RNA interfering molecule, chemically synthesized short hairpin RNA against TS (shTS), and 5-FU on the growth of human colorectal cancer cell (DLD-1) both in vitro and in vivo. The combined treatment of both shTS with 5-FU substantially inhibited cell proliferation in vitro. For in vivo treatments, the combined treatment of metronomic S-1 dosing with intravenously injected polyethylene glycol (PEG)-coated shTS-lipoplex significantly suppressed tumor growth, compared to a single treatment of either S-1 or PEG-coated shTS-lipoplex. In addition, the combined treatment increased the proportion of apoptotic cells in the DLD-1 tumor tissue. Our results suggest that metronomic S-1 dosing combined with TS silencing might represent an emerging therapeutic strategy for the treatment of patients with advanced CRC.


Modulation of antitumor immunity contributes to the enhanced therapeutic efficacy of liposomal oxaliplatin in mouse model

Immune modulation of the tumor microenvironment has been reported to participate in the therapeutic efficacy of many chemotherapeutic agents. Recently, we reported that liposomal encapsulation of oxaliplatin (l-OHP) within PEGylated liposomes conferred a superior antitumor efficacy to free l-OHP in murine colorectal carcinoma-bearing mice through permitting preferential accumulation of the encapsulated drug within tumor tissue. However, the contribution of the immune-modulatory properties of liposomal l-OHP and/or free l-OHP to the overall antitumor efficacy was not elucidated. In the present study, therefore, we investigated the effect of liposomal encapsulation of l-OHP within PEGylated liposomes on the antitumor immunity in both immunocompetent and immunodeficient mice. Liposomal l-OHP significantly suppressed the growth of tumors implanted in immunocompetent mice, but not in immunodeficient mice. In immunocompetent mice, liposomal l-OHP increased the tumor MHC-1 level and preserved antitumor immunity through decreasing the number of immune suppressor cells, including regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages, which collectively suppress CD8(+) T cell-mediated tumor cells killing. In contrast, free l-OHP ruined antitumor immunity. These results suggest that the antitumor efficacy of liposomal l-OHP is attributed, on the one hand, to its immunomodulatory effect on tumor immune microenvironment that is superior to that of free l-OHP, and on the other hand, to its direct cytotoxic effect on tumor cells.


Co-administration of liposomal l-OHP and PEGylated TS shRNA-lipoplex: A novel approach to enhance anti-tumor efficacy and reduce the immunogenic response to RNAi molecules

Many therapeutic strategies have been applied in efforts to conquer the development and/or progression of cancer. The combination of chemotherapy and an RNAi-based approach has proven to be an efficient anticancer therapy. However, the feasibility of such a therapeutic strategy has been substantially restricted either by the failure to achieve the efficient delivery of RNAi molecules to tumor tissue or by the immunostimulatory response triggered by RNAi molecules. In this study, therefore, we intended to investigate the efficacy of using liposomal oxaliplatin (liposomal l-OHP) to guarantee the efficient delivery of RNAi molecules, namely shRNA against thymidylate synthase (TS shRNA) complexed with cationic liposome (TS shRNA-lipoplex), to solid tumors, and to suppress the immunostimulatory effect of RNAi molecules, TS shRNA, following intravenous administration. Herein, we describe how liposomal l-OHP enhanced the intra-tumor accumulation of TS shRNA-lipoplex and significantly reduced the immunostimulatory response triggered by TS shRNA. Consequently, such enhanced accumulation of TS shRNA-lipoplex along with the cytotoxic effect of liposomal l-OHP led to a remarkable tumor growth suppression (compared to mono-therapy) following systemic administration. Our results, therefore, may have important implications for the provision of a safer and more applicable combination therapy of RNAi molecules and anti-cancer agents that can produce a more reliable anti-tumor effect.


Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, 1-anti-trypsin, 1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 M of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.


Preparation of an ultrafine rebamipide ophthalmic suspension with high transparency

A 2% commercially available, milky-white, rebamipide micro-particle suspension is used to treat dry eyes, and it causes short-term blurring of the patient's vision. In the current study, to improve the transparency of a rebamipide suspension, we attempted to obtain a clear rebamipide suspension by transforming the rebamipide particles to an ultrafine state. In the initial few efforts, various rebamipide suspensions were prepared using a neutralizing crystallization method with additives, but the suspensions retained their opaque quality. However, as a consequence of several critical improvements in the neutralizing crystallization methods such as selection of additives for crystallization, process parameters during crystallization, the dispersion method, and dialysis, we obtained an ultrafine rebamipide suspension (2%) that was highly transparent (transmittance at 640 nm: 59%). The particle size and transparency demonstrated the fewest level of changes at 25°C after 3 years, compared to initial levels. During that period, no obvious particle sedimentation was observed. The administration of this ultrafine rebamipide suspension (2%) increased the conjunctival mucin, which was comparable to the commercially available micro-particle suspension (2%). The corneal and conjunctival concentration of rebamipide following ocular administration of the ultrafine suspension was slightly higher than that of the micro-particle suspension. The ultrafine rebamipide suspension (eye-drop formulation) with a highly transparent ophthalmic clearness should improve a patient's QOL by preventing even a shortened period of blurred vision.


Ganglioside inserted into PEGylated liposome attenuates anti-PEG immunity

Despite the clinical introduction of a vast number of polyethylene glycol (PEG)-conjugated therapeutics, conjugated PEG is also known for an unfortunate inclination toward immunogenicity. Immunogenicity of PEG, manifested by the robust production of anti-PEG IgM, is known to compromise the therapeutic efficacy and/or reduce the tolerance of PEGylated therapeutics. In the present study, we inserted ganglioside into the membrane of PEGylated liposome (PL) to prepare ganglioside-modified PEGylated liposomes (G-PL), and investigated its efficacy in attenuating the anti-PEG IgM response against PL. A single intravenous injection of G-PL significantly attenuated the anti-PEG IgM production, compared with that of naïve PL. In addition, pretreatment with G-PL substantially alleviated the anti-PEG IgM response elicited by a subsequent dose of PL, presumably via inducing B cell tolerance, and as a consequence, this modification abrogated/attenuated the incidence of the rapid clearance of subsequently administrated PL. These results indicate that incorporating gangliosides in PEGylated liposome membrane not only prevents the immunogenicity of PEG but also induces the tolerance of B cells to subsequent doses of the immunogenic PL. Consequently, liposomal membrane modification with ganglioside might represent a promising approach to attenuating the immunogenicity of PEGylated liposomes while preserving their therapeutic efficacy, particularly upon repeated administration.


Improvement of intratumor microdistribution of PEGylated liposome via tumor priming by metronomic S-1 dosing

The efficient delivery of nanocarrier-based cancer therapeutics into tumor tissue is problematic. Structural abnormalities, tumor vasculature heterogeneity, and elevated intratumor pressure impose barriers against the preferential accumulation of nanocarrier-based cancer therapeutics within tumor tissues and, consequently, compromise their therapeutic efficacy. Recently, we have reported that metronomic S-1, orally available tegafur formulation, dosing synergistically augmented the therapeutic efficacy of oxaliplatin (l-OHP)-containing PEGylated liposome without increasing the toxicity in animal model. However, the exact mechanism behind such synergistic effect was not fully elucidated. In this study, therefore, we tried to shed the light on the contributions of metronomic S-1 dosing to the enhanced accumulation and/or spatial distribution of PEGylated liposome within tumor tissue. Tumor priming with metronomic S-1 treatment induced a potent apoptotic response against both angiogenic endothelial cells and tumor cells adjacent to tumor blood vessels, resulting in enhanced tumor blood flow via transient normalization of tumor vasculature, along with alleviation of intratumor pressure. Such a change in the tumor microenvironment imparted by S-1 treatment allows efficient delivery of PEGylated liposome to tumor tissue and permits their deep penetration/distribution into the tumor mass. Such a priming effect of S-1 dosing can be exploited as a promising strategy to enhance the therapeutic efficacy of nanocarrier-based cancer therapeutics suffering from inadequate/heterogeneous delivery to tumor tissues.


Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

Biochemical and Biophysical Research Communications


Systemically administered RNAi molecule sensitizes malignant pleural mesothelioma cells to pemetrexed therapy

Pemetrexed (PMX) is a key drug for the management of malignant pleural mesothelioma (MPM). However, its therapeutic efficacy is cruelly restricted in many clinical settings by the overexpression of thymidylate synthase (TS) gene. Recently, we emphasized the efficacy of locally administered shRNA designed against TS gene in enhancing the cytotoxic effect of PMX against orthotopically implanted MPM cells in tumor xenograft tumor model. Herein, we explored the efficiency of systemic, rather than local, delivery of TS RNAi molecule in sensitizing MPM cells to the cytotoxic effect of PMX. We here designed a PEG-coated TS shRNA-lipoplex (PEG-coated TS shRNA-lipoplex) for systemic injection. PEG modification efficiently delivered TS shRNA in the lipoplex to tumor tissue following intravenous administration as indicated by a significant suppression of TS expression level in tumor tissue. In addition, the combined treatment of PMX with systemic injection of PEG-coated TS shRNA-lipoplex exerted a potent antitumor activity in a s.c. xenograft tumor model, compared to a single treatment with either PMX or PEG-coated TS shRNA-lipoplex. Metastasis, or the spread, of mesothelioma substantially dedicates the effectiveness of treatment options. The systemic, in addition to local, delivery of tumor targeted anti-TS RNAi system we propose in this study might be an effective option to extend the clinical utility of PMX in treating malignant mesothelioma.


Hydrodynamic tail vein injection as a simple tool for yielding extended transgene expression in solid tumors.

Hydrodynamic tail vein injection was considered an in vivo transfection method that yields a higher level of gene expression mainly in the liver. This method has been applied to cancer gene therapy targeting both hepatic and non-hepatic cancers. However, intratumor transgene expression in non-hepatic tumors has not been well studied. In this study, we showed an extended transgene expression of -galactosidase (LacZ), a nonsecretory protein, in a subcutaneously implanted murine solid tumor following the hydrodynamic injection of plasmid DNA (LacZ pDNA). Our result may indicate that the hydrodynamic injection method is a powerful tool that can be used to gain transgene expression not only in the liver but also in solid tumors.


Lidocaine self-sacrificially improves the skin permeation of the acidic and poorly water-soluble drug etodolac via its transformation into an ionic liquid

Poor transdermal penetration of active pharmaceutical ingredients (APIs) impairs both bioavailability and therapeutic benefits and is a major challenge in the development of transdermal drug delivery systems. Here, we transformed a poorly water-soluble drug, etodolac, into an ionic liquid in order to improve its hydrophobicity, hydrophilicity and skin permeability. The ionic liquid was prepared by mixing etodolac with lidocaine (1:1, mol/mol). Both the free drug and the transformed ionic liquid were characterized by differential scanning colorimetry (DSC), infrared spectroscopy (IR), and saturation concentration measurements. In addition, in vitro skin-permeation testing was carried out via an ionic liquid-containing patch (Etoreat patch). The lidocaine and etodolac in ionic liquid form led to a relatively lower melting point than either lidocaine or etodolac alone, and this improved the lipophilicity/hydrophilicity of etodolac. In vitro skin-permeation testing demonstrated that the Etoreat patch significantly increased the skin permeation of etodolac (9.3-fold) compared with an etodolac alone patch, although an Etoreat patch did not increase the skin permeation of lidocaine, which was consistent with the results when using a lidocaine alone patch. Lidocaine appeared to self-sacrificially improve the skin permeation of etodolac via its transformation into an ionic liquid. The data suggest that ionic liquids composed of approved drugs may substantially expand the formulation preparation method to meet the challenges of drugs which are characterized by poor rates of transdermal absorption.

European Journal of Pharmaceutics and Biopharmaceutics


Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model

Malignant pleural mesothelioma (MPM) is an incurable cancer with an increasing incidence. Currently, pemetrexed (PMX)-based chemotherapy is the mainstay of chemotherapy for MPM, however, the outcome of PMX-based chemotherapy in patients with MPM is dismal. RNA interference (RNAi) technology has been considered as an effective tool to substantially enhance the therapeutic efficacy of chemotherapeutic agents in many preclinical and clinical settings. In this study, therefore, we investigated whether non-viral anti-thymidylate synthase RNAi embedded liposome (TS shRNA lipoplex) would effectively guide the downregulation of TS in human malignant mesothelioma MSTO-211H cells. Consequently, it enhanced the antitumor effect of PMX both in vitro and in vivo. TS shRNA effectively enhanced the in vitro cell growth inhibition upon treatment with PMX via downregulating TS expression in the MSTO-211H cell line. In in vivo orthotopic tumor model, the combined treatment of PMX and TS shRNA lipoplex efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged mouse survival, compared to each single treatment. Our findings emphasize the pivotal relevance of RNAi as an effective tool for increasing the therapeutic efficacy of PMX, a cornerstone in the treatment regimens of MPM, and thereby, raising the possibility for the development of a novel therapeutic strategy, combination therapy of TS-shRNA and PMX, that can surpass many of the currently applied, but less effective, therapeutic regimens against lethal MPM.


Hepatic tumor metastases cause enhanced PEGylated liposome uptake by Kupffer cells

Kupffer cells in livers bearing tumor metastases were found to have promoted tumor invasion and exacerbated the metastasis. This implies that the function of Kupffer cells might differ between animals bearing hepatic metastases and those that are healthy. Kupffer cells are considered responsible for the accumulation of liposomes in the liver. In this study, we hypothesized that the alteration in the function of Kupffer cells by hepatic metastasis would also affect the biodistribution of liposomes following intravenous administration. The hepatic accumulation and the blood concentration of PEGylated liposomes were compared between healthy mice and tumor-bearing mice. We noted that hepatic accumulation and elimination from the blood were significantly accelerated in tumor-bearing mice, indicating that our hypothesis was correct. In the tumor-bearing mice, the proportion of Kupffer cells taking up liposomes was significantly increased. Intravenous injection of oxaliplatin (l-OHP) containing PEGylated liposomes decreased the fraction of Kupffer cells, but this administration caused no injury to the hepatocytes. These results suggest that PEGylated liposomes containing l-OHP may have the potential to treat metastatic hepatic cancer-not only via the direct killing of the cancer cells but also via a reduction in tumor-supportive Kupffer cells.


Gene silencing using 4'-thioDNA as an artificial template to synthesize short-hairpin RNA without inducing a detectable innate immune response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4'-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4'-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4'-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.


Encapsulation in a rapid-release liposomal formulation enhances the anti-tumor efficacy of pemetrexed in a murine solid mesothelioma-xenograft model

We recently developed a PEG-coated liposome encapsulating the anti-folate drug pemetrexed (PMX). Such liposomal formulations have shown potent cytotoxic effects against malignant pleural mesothelioma (MPM) cells in vitro. In the present study, we investigated the pharmacokinetics, bio-distribution and in vivo anti-tumor efficacy of two liposomal PMX formulations with different drug release rates in a murine mesothelioma-xenograft model. Liposomes with different PMX release rates were prepared via manipulating liposomal membrane fluidity through incorporating either a solid-phase (HSPC) or a fluid-phase (POPC) phospholipid. Both liposomal PMX formulations showed prolonged plasma pharmacokinetics and were accumulated to a similar extent in tumors and other tissues, presumably, due to surface modification with polyethylene glycol (PEG). In a murine mesothelioma-xenograft model, interestingly, PMX encapsulated in a fast-release POPC liposome produced superior tumor growth suppression compared with either free PMX or PMX encapsulated in a slow-release HSPC liposome. Such in vivo anti-tumor efficacy was accomplished mainly by a potent induction of apoptosis within tumor tissue by the released PMX from POPC liposomes. Our results clearly emphasize the therapeutic efficacy of liposomal PMX over free PMX in conquering aggressive solid tumors such as malignant mesothelioma. A guarantee of the targeted delivery of PMX to tumor cells helps overcome some of the major shortcomings encountered with the use of free PMX.

European Journal of Pharmaceutical Sciences


Intra-tumor distribution of PEGylated liposome upon repeated injection: No possession by prior dose

Liposomes have proven to be a viable means for the delivery of chemotherapeutic agents to solid tumors. However, significant variability has been detected in their intra-tumor accumulation and distribution, resulting in compromised therapeutic outcomes. We recently examined the intra-tumor accumulation and distribution of weekly sequentially administered oxaliplatin (l-OHP)-containing PEGylated liposomes. In that study, the first and second doses of l-OHP-containing PEGylated liposomes were distributed diversely and broadly within tumor tissues, resulting in a potent anti-tumor efficacy. However, little is known about the mechanism underlying such a diverse and broad liposome distribution. Therefore, in the present study, we investigated the influence of dosage interval on the intra-tumor accumulation and distribution of "empty" PEGylated liposomes. Intra-tumor distribution of sequentially administered "empty" PEGylated liposomes was altered in a dosing interval-dependent manner. In addition, the intra-tumor distribution pattern was closely related to the chronological alteration of tumor blood flow as well as vascular permeability in the growing tumor tissue. These results suggest that the sequential administrations of PEGylated liposomes in well-spaced intervals might allow the distribution to different areas and enhance the total bulk accumulation within tumor tissue, resulting in better therapeutic efficacy of the encapsulated payload. This study may provide useful information for a better design of therapeutic regimens involving multiple administrations of nanocarrier drug delivery systems.


Advanced therapeutic approach for the treatment of malignant pleural mesothelioma via the intrapleural administration of liposomal pemetrexed

Malignant pleural mesothelioma (MPM) is an aggressive cancer that proliferates in the pleural cavity. Pemetrexed (PMX) in combination with cisplatin is currently the approved standard care for MPM, but a dismal response rate persists. Recently, we prepared various liposomal PMX formulations using different lipid compositions and evaluated their in vitro cytotoxicity against human mesothelioma cells (MSTO-211H). In the present study, we investigated the in vivo therapeutic effect of our liposomal PMX formulations using an orthotopic MPM tumor mouse model. PMX encapsulated within either cholesterol-containing (PMX/Chol CL) or cholesterol-free (PMX/Non-Chol CL) cationic liposome was intrapleurally injected into tumor-bearing mice. PMX encapsulated in cholesterol-free liposomes (PMX/Non-Chol CL) drastically inhibited the tumor growth in the pleural cavity, while free PMX and PMX encapsulated in cholesterol-containing liposomes (PMX/Chol CL) barely inhibited the tumor growth. The enhanced in vivo anti-tumor efficacy of PMX/Non-Chol CL was credited, on the one hand, for prolonging the retention of cationic liposomes in the pleural cavity via their electrostatic interaction with the negatively charged membranes of tumor cells, but on the other hand, it was charged with contributing to a higher drug release from the "fluid" liposomal membrane following intrapleural administration. This therapeutic strategy of direct intrapleural administration of liposomal PMX, along with the great advances in CL-guided therapeutics, might be a promising therapeutic approach to conquering the poor prognosis for MPM.


Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells

The accelerated blood clearance (ABC) phenomenon makes it crucial to use PEGylated liposomes and micelles to deliver drugs. The ABC phenomenon is an immune response against an initial dose of PEGylated liposome, which causes subsequent doses to be rapidly cleared by macrophages in the liver. We recently found that in the early phase of the ABC phenomenon, subsequent doses of PEGylated liposomes were associated with splenic marginal zone (MZ)-B cells and were transported from the MZ to the follicle (FO). In this study, we investigated the underlying mechanisms behind the association of subsequent doses of PEGylated liposomes with MZ-B cells in the spleen. Serum factors, anti-PEG IgM and complement system, were crucial to the association of PEGylated liposomes with MZ-B cells, while the sensitization of MZ-B cells by the first dose of PEGylated liposomes was not significant. It was the complement receptors (CRs) on the MZ-B cells, rather than either the PEG-specific B-cell receptors or the IgM Fc receptors, that were the main contributors to the association between PEGylated liposomes and MZ-B cells. It appeared that anti-PEG IgM would bind to PEGylated liposomes and causes subsequent complement activation, resulting in the formation of immune complexes of PEGylated liposome-anti-PEG IgM-complement. The MZ-B cells then recognized these immune complexes via their CRs. Such an association via CRs might have triggered the transport of the immune complex by MZ-B cells to the FO in the spleen. The information obtained in this study might be useful in the development of an efficient antigen delivery system to usher PEGylated nanoparticles into FO dendritic cells.


Comprehensive analysis of PEGylated liposome-asscociated proteins relating to the accelerated blood clearance phenomenon by combination with shotgun analysis and conventional methods

PEGylated liposome, sterically stabilized by polyethylene glycol (PEG), results in reduced recognition of the liposome by the mononuclear phagocyte system. Recently, we reported regarding the accelerated blood clearance (ABC) phenomenon that PEGylated liposome is cleared very rapidly from blood circulation upon repeated injection. Anti-PEG IgM production and subsequent complement activation were crucial in causing the ABC phenomenon. However, there still remains the possibility that unknown plasma factors might affect the fate of PEGylated liposome that is subjected to the ABC phenomenon. A label-free approach to shotgun analysis is a great tool for characterizing proteins in a biological system. In this study, therefore, a shotgun analysis was employed to identify plasma protein bound on PEGylated liposome after the ABC phenomenon was induced in the mouse model. The analysis revealed that immunoglobulin and complement components (C1 and C3) are the major proteins. Subsequent analysis with enzyme-linked immunosorbent assay and Western blotting showed that the immunoglobulin was IgM and that the complement system was mainly activated via an anti-PEG IgM-mediated classical pathway. These results support our earlier assumptions-anti-PEG IgM and complement activation were the major causes of the ABC phenomenon. Our proposed analytical strategy would be expected to provide useful information for the development and design of the nanocarrier drug delivery system.


Anti-PEG IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein

Limited therapeutic efficacy of polyethylene glycol-conjugated (PEGylated) protein drugs has been recently reported in animals and human following repeat injections. Since there are reports that an accelerated blood clearance (ABC) phenomenon is caused by repeated injection of PEGylated liposome, there is an assumption that PEGylated proteins lose their long circulating property when they are injected repeatedly due to the induction of anti-PEG antibody. Although induction of anti-PEG antibody by PEGylated protein has been reported, there is little evidence of accelerated blood clearance of PEGylated protein upon repeated injection. Herein, we investigated the blood concentration of PEGylated ovalbumin (PEG-OVA), a model PEGylated protein, upon its repeated injection. A single intravenous administration of PEG-OVA elicited an anti-PEG IgM response but not anti-PEG IgG response, while the administration did not elicit antibody against OVA. At 24 h postinjection of test PEG-OVA, although control mice showed 41.6% dose of PEG-OVA in blood, the mice pretreated with PEG-OVA showed rapid clearance of test PEG-OVA from blood and undetectable level of PEG-OVA. Interestingly, the anti-PEG IgM induced by PEGylated liposome did not affect the blood concentration of subsequent dose of PEG-OVA. Our result suggests that anti-PEG IgM is a major contributor to the accelerated blood clearance of PEG-conjugated protein, but the presence of anti-PEG IgM in blood circulation does not necessarily affect circulating property of entire PEGylated materials.


Synthesis of DNA fragments containing 2-deoxy-4-selenonucleoside units using DNA polymerases: comparison of dNTPs with O, S and Se at the 4-position in replication Org

4'-SelenoDNA fragments were synthesized for the first time using 4'-selenothymidine triphosphate (SeTTP) by taking advantage of its bioequivalence against DNA polymerases. DNA fragments each with a homologous element (O, S or Se) at the 4'-position of the thymidine units were effectively amplified using KOD Dash DNA polymerase.


Relationship between the concentration of anti-polyethylene glycol (PEG) in immunoglobulin M (IgM) and the intensity of the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes in mice

PEGylation, which is the surface modification of nanocarriers with polyethylene glycol (PEG), has increased the circulation time and reduced the immunogenic responses to nanocarriers. However, many reports have demonstrated that the intravenous injection of sterically stabilized PEGylated liposome (SL) causes an accelerated blood clearance (ABC) of subsequent doses via anti-PEG immunoglobulin M (IgM)-mediated complement activation. In the present study, the relationships between serum anti-PEG IgM concentration, the intensity of complement activation and the hepatic clearance of SL were quantitatively investigated for their role in the ABC phenomenon. Interestingly, with increasing serum anti-PEG IgM concentrations, the intensity of complement activation increased linearly, while the intensity of the hepatic clearance of SL was increased and then saturated. In addition, only 15-17% of anti-PEG IgM in blood circulation induced by SL at different doses was associated with a second dose SL. The present results indicate that it is the hepatic uptake of SL that is the limiting step in the ABC phenomenon, rather than the association of anti-PEG IgM to the SL and a subsequent complement activation.


Liposomal pemetrexed: Formulation, characterization and in vitro cytotoxicity studies for effective management of malignant pleural mesothelioma

Pemetrexed (PMX) is a newly developed multi-targeted anti-folate with promising clinical activity in many solid tumors including malignant pleural mesothelioma (MPM). However, PMX does not show sufficient anti-tumor activity in vivo when used alone either due to inefficient delivery of adequate concentrations to tumor tissue or dose-limiting side effects. In order to overcome these problems and to achieve potent anti-tumor activity, PMX was encapsulated into a liposomal delivery system. In the present study, various formulations of liposomal PMX were prepared. The effect of formulation parameters on the encapsulation efficiency of PMX within liposomes was evaluated. In addition, the influence of drug release rate on the in vitro cytotoxicity was investigated. Encapsulation of PMX within liposomes was remarkably increased by the incorporation of cholesterol within liposomal membranes and by increasing the total lipid concentration. Encapsulation efficiency was found to be unaffected by the type of phospholipid used or the inclusion of a cation lipid, DC-6-14. Interestingly, encapsulation of PMX within "fluid" liposomes was found to allow efficient release of PMX from liposomes resulting in a potent in vitro cytotoxicity against MPM MSTO-211H cell line. On the other hand, entrapment of PMX within "solid" liposomes substantially hindered PMX release from liposomes, and thus PMX failed to exert any in vitro cytotoxicity. These results suggest that encapsulation of PMX within "fluid" liposomes might represent a novel strategy to enhance the therapeutic efficacy of PMX while minimizing the side effect encountered by the non selective delivery of free PMX to various body tissues.


Influence of dose and animal species on accelerated blood clearance of PEGylated liposomal doxorubicin

We recently demonstrated that Doxil loses its long-circulating properties when injected repeatedly at doses below 2 mg/m(2) in dogs. In studies using other animal species, PEGylated liposomal doxorubicin has been reported not to induce the accelerated blood clearance (ABC) phenomenon. We investigated the issue of whether Doxil can elicit the ABC phenomenon in several species. In minipigs, the ABC phenomenon was induced at 2 mg/m(2). In other animal species, the ABC phenomenon was not observed at higher doses (>2 mg/m(2)), but was observed at much lower doses (0.2 mg/m(2)). The pharmacokinetic profile of a second dose of Doxil reflected the circulating anti-PEG IgM level induced by the first dose. The ABC phenomenon was not observed at the clinically recommended DXR dose (20 mg/m(2)) in any animal species. These results indicate that Doxil can cause the ABC phenomenon in all animals tested, the extent of induction was dependent on the first dose of Doxil, and a higher Doxil dose lessened the ABC phenomenon. The current study results suggest that a careful study design including selection of animal species is important for preclinical studies using PEGylated liposomal formulations even if they contain anticancer drugs that suppress the host immune response.


Chemistry, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA, a novel hybrid type of chemically modified RNA.

We report the synthesis, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA (MOE-SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE-SRNA showed a moderate improvement of Tm value (+3.4 °C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE-SRNA relative to 2'-O-methoxyethylRNA (MOERNA), 2'-O-methyl-4'-thioRNA (Me-SRNA), 2'-O-methylRNA (MeRNA), 4'-thioRNA (SRNA), and natural RNA revealed that MOE-SRNA had the highest stability (t1/2 >48 h in human plasma). Because of the favorable properties of MOE-SRNA, we evaluated its in vitro and in vivo potencies as an anti-microRNA oligonucleotide against miR-21. Although the in vitro potency of MOE-SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).


B cell-intrinsic toll-like receptor 7 is responsible for the enhanced anti-PEG IgM production following injection of siRNA-containing PEGylated lipoplex in mice

Recently, we reported that immunostimulatory siRNA-containing PEGylated lipoplex (PEGylated siRNA-lipoplex) activates the immune system, resulting in the enhanced production of anti-PEG IgM. However, the enhancing mechanism upon anti-PEG IgM production has not been fully elucidated. In this study, we employed toll-like receptor 7 knock out (TLR7 KO) mice, and showed how PEGylated siRNA-lipoplex activates the innate immune system through TLR7 and consequently enhances anti-PEG IgM production. In addition, we showed that SCID mice reconstituted with TLR7-deficient B cells failed to enhance anti-PEG IgM production following the injection of PEGylated siRNA-lipoplex, but that SCID mice reconstituted with wild type B cells did enhance anti-PEG IgM production. These results suggest that immune activation via B cell-intrinsic TLR7, but not other TLR7-expressing cells, contributes predominantly to an enhanced anti-PEG IgM production in response to the intravenous injection of PEGylated siRNA-lipoplexes. A strategy to evade B cell-intrinsic TLR7 activation by siRNA, such as chemical modification, may overcome immunological barriers to PEGylated liposome-based siRNA therapeutics.


Activation of TLR9 by incorporated pDNA within PEG-coated lipoplex enhances anti-PEG IgM production

Cationic liposome represents a promising alternative to viral vectors for the delivery of therapeutic genes. For in vivo use, surface modification of the liposome with polyethylene glycol (PEG) is frequently applied to achieve gene-expression in the targeted tissue. However, we have reported that PEG-coated liposomes have induced anti-PEG IgM, which has caused subsequent doses of PEG-coated liposome to be rapidly cleared from blood circulation, and the complexation of pDNA electrostatically associated with liposome surface has enhanced this antibody response. In this study, we investigated how a Toll-like receptor (TLR) might enhance anti-PEG IgM production. PEG-coated pDNA-lipoplex (PDCL) was injected into either wild type, MyD88 (all TLR adaptor protein, independent of TLR3) knock out (KO) or TLR9 KO mice, and the anti-PEG IgM production levels were detected. Attenuated anti-PEG IgM production following the injection of PDCL was observed in both MyD88 and TLR9 KO mice compared to wild type mice, probably due to the abolished induction of cytokines in both MyD88 and TLR9 KO mice. Our results suggest that TLR, exclusively TLR9, signaling plays a potential role in the enhanced anti-PEG IgM production following the injection of PDCL. This result may have important implications for the design and development of an efficient PEG-coated non-viral gene vector.


Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs--HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG2000 conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH2CH2) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics.


Novel Submicronized Rebamipide Liquid with Moderate Viscosity: Significant Effects on Oral Mucositis in Animal Models

This study aimed at developing a novel rebamipide liquid for an effective treatment of oral mucositis. The healing effects of a variety of liquids comprising submicronized rebamipide crystals were investigated using a rat cauterization-induced oral ulcer model. Whereas 2% rebamipide liquid comprising micro-crystals did not exhibit significant curative effect, 2% rebamipide liquids comprising submicronized crystals with moderate viscosities exhibited healing effects following intra-oral administration. The 2% and 4% optimized rebamipide liquids showed significant healing effects in the rat oral ulcer model (p<0.01). In addition, in the rat radiation-induced glossitis model, whereby the injury was caused to the tongue by exposing only around the rat's snout to a 15 Gy of X-irradiation, the 2% optimized rebamipide liquid significantly reduced the percent area of ulcerated injury (p<0.05). In conclusion, the submicronized rebamipide liquid with moderate viscosity following intra-oral administration showed better both healing effect in the rat oral ulcer model and preventive effect in the rat irradiation-induced glossitis model.


Sequential treatment of oxaliplatin-containing PEGylated liposome together with S-1 improves intratumor distribution of subsequent doses of oxaliplatin-containing PEGylated liposome

We recently reported that combination therapy with metronomic S-1 dosing and oxaliplatin (l-OHP)-containing PEGylated liposomes improved antitumor activity in a murine colorectal tumor model. However, little is known about the mechanism underlying such improved therapeutic efficacy. Here we investigated the impact of combined treatment on biodistribution, tumor accumulation and intratumor distribution of test PEGylated liposomes and on the structure of tumor vasculature in a solid tumor. The combined treatment clearly enhanced tumor accumulation and intratumor distribution of a subsequent test dose of PEGylated liposome as a result of on the one hand prolonging blood circulation of test liposome and on the other hand the alteration in tumor microenvironment. The l-OHP-containing PEGylated liposomes contributed predominantly to the enhanced tumor accumulation and altered tumor distribution of test liposome. On the other hand, metronomic S-1 dosing contributed to the altered tumor distribution but not the tumor accumulation of test liposome. The antitumor effect of the combined treatment, reflected by the proportion of apoptotic cells in the tumor, was approximately equally accounted for by each of the two treatments, leading to a roughly additive effect. In conclusion, 1-OHP-containing PEGylated liposome together with S-1 enhanced intratumor influx, leading to improved antitumor activity of subsequently injected 1-OHP-containing PEGylated liposomes and/or S-1. This strategy we propose, which is clinically applicable, may overcome the problems related to the use of EPR effect-based nanocarrier systems.

European Journal of Pharmaceutics and Biopharmaceutics


Application of polyglycerol-coating to pDNA lipoplex for the evasion of the accelerated blood clearance (ABC) phenomenon in nucleic acid delivery.

Cationic liposomes (CLs) have shown promise as nonviral delivery systems. To achieve in vivo stability and long circulation, most liposomes are modified with hydrophilic polymer polyethylene glycol (PEG). However, we have reported that repeated administration of PEG-coated CLs containing plasmid DNA (pDNA; PEGylated lipoplexes) induces what is referred to as "the accelerated blood clearance (ABC) phenomenon" and, consequently, subsequently administered lipoplexes lose their prolonged circulation characteristics. Anti-PEG IgM produced in response to the first dose of PEG-coated pDNA-lipoplexes (PEG-DCL) has proven to be a major cause of the ABC phenomenon. In this study, to evade and/or attenuate this unexpected immune response, we modified the surface of a lipoplex with polyglycerol (PG)-derived lipid. The PG-coated pDNA-lipoplex (PG-DCL) attenuated the production of anti-polymer IgM, whereas PEG-coated pDNA-lipoplex (PEG-DCL) did not. In addition, a second dose of PG-DCL maintained the accumulation level in the tumor tissue of a tumor-bearing mouse model, comparable to that of the first dose, whereas the tumor accumulation level of a second dose of PEG-DCL was significantly compromised, compared with the first dose of PEG-DCL. Our results indicate that surface modification of lipoplex with PG represents a viable means for the attenuation, and/or evasion, of the ABC phenomenon that is encountered upon repeated administrations of nucleic acids containing PEG-coated nanocarriers.


Ex-Vivo/In-Vitro Anti-Polyethylene Glycol (PEG) IgM Production From Murine Splenic B Cells Stimulated by PEGylated Liposome

We have reported that PEGylated liposomes lose their long-circulating properties when injected twice into the same animal within a certain interval (the accelerated blood clearance (ABC) phenomenon). We assumed that this phenomenon was triggered via the abundant secretion of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) in response to the first dose of PEGylated liposomes and that the spleen played an important role in the production of anti-PEG IgM. However, no direct evidence has yet confirmed this suspicion. In the current study, we verified, both in vitro and ex vivo, that spleen cells are indeed responsible for the production of anti-PEG IgM in response to PEGylated liposomes. In this study, spleen cells obtained from either naïve mice or mice pre-treated with PEGylated liposomes induced the production of anti-PEG IgM in a dose- and time-dependent manner, upon incubation with PEGylated liposomes. In addition, we confirmed that among the different fractions of splenic B cells, IgM-positive B cells, rather than CD45R-positive or CD19-positive splenic B cells, which are presumed to be the marginal zone B (MZB) cells, are the major cells producing anti-PEG IgM in the response to stimulation by PEGylated liposomes. These results may provide new insights into the mechanisms underlying the anti-PEG IgM production in response to the stimulation by PEGylated liposomes.


Establishment of an X-ray irradiation-induced glossitis model in rats: biphasic elevation of proinflammatory cytokines and chemokines

Oral mucositis is a frequent and serious side effect in patients who receive radiotherapy for head and neck cancer. The purpose of this study was to develop a noninvasive and quantitative model of oral mucositis in rats, investigate the pathophysiology, and evaluate the efficacy of pharmacological interventions. Rats received a single dose of 15 Gy of X-rays to the snout after shielding of the remainder of the rat body with lead plates to protect the body from irradiation (day 0). After irradiation, the macroscopic area of tongue injury gradually increased. The total area of injury and the ulcer-like area reached a maximum on day 7 and then gradually decreased until disappearance on day 28. Expression of proinflammatory cytokines and chemokines occurred transiently within 1-4 hours after irradiation and returned to a normal level at 24 hours. This expression was again observed from days 3 to 5 and increased significantly on day 7, which approximately coincided with the histologic severity of tissue damage. Subcutaneous administration of palifermin at 3 mg/kg per day for 3 consecutive days before irradiation completely prevented ulcer formation in this model. In conclusion, we established a novel model of glossitis in rats, induced by X-ray irradiation, in which biphasic elevations of expression of proinflammatory cytokines and chemokines could be monitored. This model is considered useful to investigate the pathophysiology of oral mucositis and evaluate the preventive effect of pharmacological interventions on oral mucositis induced by X-ray irradiation.

The Journal of Pharmacology and Experimental Therapeutics


Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration

The accelerated blood clearance (ABC) phenomenon accounts for the rapid systemic clearance of PEGylated nanocarriers upon repeated administrations. IgM production against the polyethylene glycol (PEG) coating in PEGylated liposomes is now known to be responsible for such unexpected pharmacokinetical alterations. The ABC phenomenon poses a remarkable clinical challenge by reducing the therapeutic efficacy of encapsulated drugs and causing harmful effects due to the altered tissue distribution pattern of the drugs. In this study, we investigated the in vivo performance of liposomes modified with polyglycerol (PG) upon repeated injection, and the in vivo therapeutic efficacy of such liposomes when they encapsulated a cytotoxic agent, doxorubicin (DXR). Repeated injection of PEG-coated liposomes in rats induced the ABC phenomenon, while repeated injection of PG-coated liposomes did not. In addition, DXR-containing PG-coated liposomes showed antitumor activity that was superior to that of free DXR and similar to that of DXR-containing PEG-coated liposomes upon repeated administration. These results indicate that polyglycerol (PG) might represent a promising alternative to PEG via enhancing the in vivo performance of liposomes by not eliciting the ABC phenomenon upon repeated administration.


The co-delivery of oxaliplatin abrogates the immunogenic response to PEGylated siRNA-lipoplex.

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.


Development of siRNA Delivery Strategy by Active Control of Tumor Microenvironment

腫瘍内微小環境の能動的制御に基づくsiRNAデリバリー技術の開発とがん治療への展開

Efficient systemic siRNA delivery to cells in the target tissue is a current critical challenge in the drug delivery field. Several studies have demonstrated that nanoparticles such as polyethylene glycol (PEG)-coated siRNA-lipoplexes may enhance the systemic delivery of siRNA to tumor. However, the disordered tumor microenvironment still poses a potential impediment with respect to the efficient delivery of PEG-coated siRNA-lipoplexes. We recently showed that metronomic S-1 dosing (daily oral administration) enhanced the accumulation of PEG-coated liposome containing anticancer drug in solid tumor tissue and thereby increased therapeutic efficacy in tumor-bearing mouse model. To extend this work, we tried to investigate the effect of metronomic S-1 dosing on the intratumoral accumulation of PEG-coated siRNA-lipoplex and, thereby, their therapeutic efficacy in solid tumor-bearing mouse model. Results showed that metronomic S-1 dosing improved systemic delivery of intravenously injected PEG-coated siRNA-lipoplexes into solid tumor tissue. In addition, the combined therapy of S-1 and PEG-coated siRNA-lipoplexes showed potent tumor growth suppressive effect. Our proposed strategy may pose a promising therapeutic one to conquer cancer progression with siRNA.<br>

Journal of the Pharmaceutical Society of Japan


Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon.

The accelerated blood clearance (ABC) phenomenon has been reported to enhance the clearance of PEGylated liposomes from the blood circulation when the liposomes are injected into the same animal repeatedly. We have shown that anti-PEG IgM production from splenic B cells is crucial in the ABC phenomenon. In this study, we describe the crucial role of marginal zone (MZ) B cells in the anti-PEG IgM production and recognition of PEGylated liposomes in the induction phase of ABC phenomenon. Suppression of the anti-PEG IgM production was correlated with the disappearance of IgM(high) cells in the MZ, particularly MZ-B cells, following cyclophosphamide (CPA)-treatment, confirming that splenic MZ-B cells are responsible for anti-PEG IgM production. The MZ-B cells stimulated by a first dose of PEGylated liposomes internalized the second dose of PEGylated liposomes in a PEG modification-dependent manner and transported the liposomes into the follicle (FO) region. To the best of our knowledge, this is the first report showing that PEGylated liposome is recognized by MZ-B cells and transported to the FO region like blood-borne antigens or immune complexes. It is likely that PEGylated liposomes are recognized as a TI-2 antigen by the first line of defense against life-threatening infections by blood-borne organisms. Our study may have implications for immunogenicity of synthesized polymer-grafted therapeutics including nanocarriers, nucleic acids and proteins.


Suppression of immune response by antigen-decorated liposomes encapsulating model agents: A novel strategy for the treatment of allergy.

A specific antigen-sensitized animal has antigen-specific immune cells that recognize the antigen. Therefore, an antigen-modified drug carrier would be recognized by the immune cells. When such a carrier encapsulates certain drugs, these drugs should be specifically delivered to the immune cells. To examine this strategy, ovalbumin (OVA) was used as model antigen, and mice were presensitized with 100 μg of OVA with Alum. For preparing OVA-modified liposomes (OVA-lipo), OVA was incubated with DSPE-PEG-NHS and resulting DSPE-PEG-OVA was inserted into liposomes. OVA-specific IgG was produced 6-fold higher by intravenous injection of OVA-lipo thrice (10 μg as OVA in each injection) in OVA-sensitized mice, than that by the injection of control liposomes, suggesting that OVA-lipo was recognized by the antigen-specific immune cells. Moreover, intra-splenic accumulation of OVA-lipo was observed in OVA-sensitized mice, but not in naive mice. To achieve the delivery of a drug to specific immune cells, OVA-lipo encapsulated low dose of doxorubicin (DOX) as a model drug (20 μg DOX/mouse, Ca. 1 mg/kg) was injected in the sensitized mice. The injection of OVA-lipo encapsulating DOX suppressed the production of IgE against OVA, suggesting that the specific delivery of the drug to immune cells responsible for OVA recognition was achieved and that these immune cells were removed by the drug treatment. This strategy would be useful for the fundamental treatment of allergy by the use of immunosuppressing agents.


Abrogation of the accelerated blood clearance phenomenon by SOXL regimen: Promise for clinical application.

We recently proposed an S-1 combined with oxaliplatin (SOXL) regimen, a combination treatment consisting of oral metronomic S-1 dosing and intravenous administration of oxaliplatin (l-OHP) containing PEGylated liposomes, which showed potent antitumor activity in vivo. PEGylated liposomes induce what is referred to as the "accelerated blood clearance (ABC) phenomenon" upon repeated administration and consequently lose their long-circulating characteristics. This phenomenon seems to pose an impediment for the clinical application and use of PEGylated liposomal formulations. In the present study, l-OHP-containing PEGylated liposomes in the SOXL regimen significantly attenuated the ABC phenomenon in a dose-dependent manner through suppression of the anti-PEG IgM response, which allowed an enhanced hepatic uptake of subsequently injected test PEGylated liposomes. In tumor-bearing mice, the abrogation of the ABC phenomenon restored intratumor accumulation of subsequently injected PEGylated liposomes. Consequently, the therapeutic efficacy of the SOXL regimen over the combination of the free form of the drugs was credited not only with the selective delivery of drugs to the tumor tissue but also with ensuring an adequate accumulation of subsequent doses within the tumor tissue. The SOXL regimen we proposed may hold promise as a safe and effective treatment regimen for advanced colorectal cancer.


Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs.

The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.


Multiple administration of PEG-coated liposomal oxaliplatin enhances its therapeutic efficacy: a possible mechanism and the potential for clinical application.

We previously developed a PEG-coated cationic liposome that enabled dual targeting delivery of oxaliplatin (l-OHP) to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation consequently elicited potent antitumor efficacy in a murine solid tumor model after 3 sequential injections. However, the probable mechanism(s) for this enhanced antitumor activity has not been fully elucidated. In the present study, therefore, the changes in tumor microenvironment induced by sequential administration of liposomal l-OHP were investigated, with emphasis on its impact to the intratumoral localization of the subsequently injected dose. In addition, the potential for anti-PEG IgM production upon repeated administration of liposomal l-OHP-containing PEGylated lipid was clearly revealed. Two sequential injections of liposomal l-OHP induced superior apoptotic activity in tumor tissue and thus resulted in broader intratumor distribution of the subsequent test dose of PEG-coated cationic liposomes, compared with a single injection of liposomal l-OHP. In addition, it was confirmed that repeated administration of liposomal l-OHP did not induce a significant anti-PEG IgM response, indicating that l-OHP encapsulated in PEG-coated liposomes was efficient in abrogating the ABC phenomenon. These results suggest that sequential treatment strategies with liposomal cytotoxic agents might be superior to mono-treatment strategies in achieving alterations in the tumor microenvironment and maintaining/restoring the pharmacokinetics of the formulation, and, therefore, would result in substantial therapeutic efficacy.


Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response.

A single intravenous administration of polyethylene glycol-coated (PEGylated) bovine serum albumin (BSA) and ovalbumin (OVA) elicited an anti-PEG immunoglobulin M (IgM) response, similar to that from PEGylated liposomes, although the administration did not elicit specific neutralizing antibodies to BSA and OVA. A cross-reactivity was observed between anti-PEG IgMs elicited by PEG-BSA and PEGylated liposomes. The anti-PEG IgM level induced by PEGylated proteins (BSA and OVA) reached the maximum at day 5 following intravenous injection. This production pattern was consistent with that induced by PEGylated liposomes. Splenectomy suppressed the anti-PEG IgM response against PEG-BSA and PEGylated liposomes. These observations relating PEG-BSA and PEGylated liposomes indicate that PEGylated proteins might promote the immune responses against PEG with a mechanism similar to that of PEGylated liposomes. In addition, a single intravenous administration of PEGylated adenovirus (PEG-Ad) also elicited an anti-PEG IgM response in a PEG-modification ratio dependent manner. To the best of our knowledge, this is the first report showing that an intravenous administration can elicit an anti-PEG IgM response against PEGylated substances. It appears that anti-PEG IgMs can be produced by the systemic administration of a PEGylated substance and may limit the efficacy of PEGylated substances such as proteins, Ad vector and nanoparticles, due to a cross-reactivity seen in some patients. The immunogenicity of PEGylated substances is usually tested against those very substances, rather than against covalently attached PEG. Our study suggests that the PEG immunogenicity of PEGylated therapeutic agents and particles merits further investigation.


Tumor type dependent vascular permeability constitutes a potential impediment to the therapeutic efficacy of liposomal oxaliplatin.

The delivery of anticancer agents to solid tumors is problematic. Nanomolecular drug carriers represent an attractive alternative strategy for efficient anticancer drug delivery to tumor tissue, because they appear to target tumors and have limited toxicity in normal tissue. However, inadequate and heterogeneous distribution of nanocarriers in tumor tissue is a major impediment for their efficient use in clinical cancer therapy. In the present study, we examined the effect of tumor type on the intratumor accumulation and distribution of polyethylene glycol (PEG)-coated liposomes using in vivo mouse models of three cancer cell lines: colon adenocarcinoma (C26), Lewis lung carcinoma (LLC), and B16BL6 melanoma (B16BL6). The tumor growth inhibition and the apoptotic response of oxaliplatin (l-OHP) encapsulated in the PEG-coated liposomes were tumor type dependent and correlated with a tendency toward tumor accumulation and intratumor distribution of PEG-coated liposome, in contrast to in vitro cytotoxicity of l-OHP. A potent antitumor effect observed in both C26 and LLC tumor-bearing mice was attributed to the enhanced extravasation with subsequent preferential accumulation of PEG-coated liposomes through tumor vasculature with high permeability. Our results suggest that the permeability of tumor vasculature constitutes a potential impediment to tumor localization and thereby to the antitumor efficacy of PEG-coated liposomal anticancer drugs.

European Journal of Pharmaceutics and Biopharmaceutics


Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes.

We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.


Improved intratumoral delivery of PEG-coated siRNA-lipoplexes by combination with metronomic S-1 dosing in a murine solid tumor model

Drug Delivery and Translational Research


Combination therapy with metronomic S-1 dosing and oxaliplatin-containing PEG-coated cationic liposomes in a murine colorectal tumor model: Synergy or antagonism?

Combination therapy with 2 or more drugs with different mechanisms of action has been considered a promising strategy for the effective treatment of advanced and metastatic cancers. However, the rational design of combination therapy represents a potential prerequisite for its effectiveness. Recently, we showed that the combination of oral metronomic S-1 dosing with oxaliplatin (l-OHP)-containing PEG-coated "neutral" liposomes exerted excellent antitumor activity. In addition, we recently designed a PEG-coated "cationic" liposome for dual-targeting delivery of l-OHP to tumor endothelial cells and tumor cells in a solid tumor. This targeted liposomal l-OHP formulation showed efficient antitumor activity in a murine tumor model, compared with l-OHP-containing PEG-coated "neutral" liposomes. In the present study, we investigated the issue of whether metronomic S-1 dosing with l-OHP-containing PEG-coated "cationic" liposomes creates synergy. Unfortunately, metronomic S-1 dosing resulted in impaired delivery of PEG-coated "cationic" liposomes into tumor tissue, presumably by decreasing the binding sites on tumor blood vessels available for the liposomes. The anticipated cytotoxic synergistic effect of the combination treatment was not achieved. Instead, the combination treatment showed lower antitumor efficacy than l-OHP-containing PEG-coated "cationic" liposomes alone. These results suggest that the combined treatment of S-1 and l-OHP-containing PEG-coated "cationic" liposomes seems to be antagonistic rather than synergistic.


Anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery.

siRNA has been touted as a therapeutic molecule against genetic diseases, which include cancers. But several challenging issues remain in order to achieve efficient systemic siRNA delivery and a sufficient therapeutic effect for siRNA in vivo. Cationic liposome shows promise as a carrier for nucleic acids, as it can selectively bind to angiogenic tumor blood vessels. In this way, anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery could be achieved in cancer therapy. In the present study, we proved our assumption by preparing various kinds of polyethylene glycol (PEG)-coated siRNA/cationic liposome complexes (siRNA-lipoplexes) and screening the avidity of these siRNA-lipoplexes upon angiogenic tumor blood vessels by means of a murine dorsal air sac (DAS) model. The lipoplex, having a lipid composition of DC-6-14/POPC/CHOL/DOPE/mPEG(2000)-DSPE=20/30/30/20/5 (molar ratio) and a charge ratio of cationic liposome and siRNA=3.81 (+/-), showed a higher binding index to newly formed blood vessels. Systemic injection with the lipoplex containing siRNA for the Argonaute2 gene (apoptosis-inducible siRNA) resulted in significant anti-tumor effect without severe side effects in mice with Lewis lung carcinoma. Our results indicate that the PEGylated cationic liposome-mediated systemic delivery of cytotoxic siRNA achieves anti-angiogenesis, resulting in the suppression of tumor growth.


A double-modulation strategy in cancer treatment with a chemotherapeutic agent and siRNA.

5-Fluorouracil (5-FU) is broadly considered the drug of choice for treating human colorectal cancer (CRC). However, 5-FU resistance, mainly caused by the overexpression of antiapoptotic proteins such as Bcl-2, often leads ultimately to treatment failure. We here investigated the effect of Bcl-2 gene silencing, using small interfering RNA (siRNA) (siBcl-2), on the efficacy of 5-FU in CRC. Transfection of siBcl-2 by a Lipofectamine2000/siRNA lipoplex effectively downregulated Bcl-2 expression in the DLD-1 cell line (a CRC), resulting in significant cell growth inhibition in vitro upon treatment with 5-FU. For in vivo treatments, S-1, an oral formulation of Tegafur (TF), a prodrug of 5-FU, was used to mimic 5-FU infusion. The combined treatment of polyethylene glycol (PEG)-coated siBcl-2-lipoplex and S-1 showed superior tumor growth suppression in a DLD-1 xenograft model, compared to each single treatment. Surprisingly, daily S-1 treatment enhanced the accumulation of PEG-coated siBcl-2-lipoplex in tumor tissue. We propose a novel double modulation strategy in cancer treatment, in which chemotherapy enhances intratumoral siRNA delivery and the delivered siRNA enhances the chemosensitivity of tumors. Combination of siRNA-containing nanocarriers with chemotherapy may compensate for the limited delivery of siRNA to tumor tissue. In addition, such modulation strategy may be considered a promising therapeutic approach to successfully managing 5-FU-resistant tumors.


Argonaute2 is a potential target for siRNA-based cancer therapy for HT1080 human fibrosarcoma.


Immunoliposomal drug delivery system targeting lectin-like oxidized low density lipoprotein receptor 1 for carotid plaque lesion in rats.

Targeted drug delivery with immunoliposomes has been applied to various in vivo animal models and is newly focused as a novel therapeutic target. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) is a potent regulator of systemic atherosclerosis, and the authors focused on its effect on carotid plaques. The authors developed a LOX1-targeted liposomal rho-kinase inhibitor and examined the therapeutic effect on carotid intimal hypertrophy in rats. LOX1-targeted rho-kinase inhibitor fasudil-containing liposomes, composed of hydrogenated soy phosphatidylcholine/cholesterol/PEG(2000)-DSPE, were prepared by conjugating anti-LOX1 antibodies on the surface and by remote loading of fasudil. Carotid intimal hypertrophy was induced by balloon injury, and the drugs were intravenously administered on Day 3 postinjury. The rats were divided into 4 groups: nontreatment, treatment with intravenous fasudil (2 mg), treatment with liposomal fasudil (2 mg), and treatment with LOX1-targeted liposomal fasudil (2 mg). The authors compared intimal hypertrophy, atherosclerotic factor, and matrix metalloproteinase-9 expression among groups. DiI-labeled LOX1-targeted liposomes were prominently observed in the lesions on Day 7 after the surgery. The intimal thickness was significantly reduced in the LOX1-targeted liposomal fasudil-treated group (mean 81.6 ± 13.9 m) compared with the other groups (no treatment 105.4 ± 16.8 m; fasudil treatment 102.4 ± 20.0 m; and liposomal fasudil treatment 102.8 ± 22.2 m; p = 0.046). Matrix metalloproteinase-9 expression was also significantly reduced in the LOX1-targeted liposomal fasudil group. Liposomes conjugated with anti-LOX1 antibody effectively reached carotid artery lesions, and liposomal rho-kinase significantly inhibited intimal hypertrophy. The new liposomal drug delivery system targeting LOX1 may become a therapeutic strategy for atherosclerotic diseases.


Agitation during lipoplex formation improves the gene knockdown effect of siRNA.

The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.


Anti-PEG IgM response against PEGylated liposomes in mice and rats.


Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA.

We recently reported that the prolonged circulation property of PEGylated cationic liposomes containing nucleic acids disappears, if the second dose is injected within a few days later, due to the production of anti-PEG IgM. This accelerated blood clearance is a concern for treating diseases which require repeated treatment with a PEGylated formulation containing nucleic acids. In this study, we investigated the effect of encapsulation of siRNA in a recently introduced PEGylated lipid nanocarrier for which the term "wrapsome" (PEGylated wrapsome, PEG-WS) was proposed as well as the sequence of the encapsulated siRNA on anti-PEG IgM production. siRNA encapsulated in PEG-WS produced little anti-PEG IgM relative to siRNA in conventional PEGylated lipoplexes. The sequence of siRNA in the PEG-WL dramatically affected the anti-PEG IgM production; a potent immune stimulatory siRNA induced a higher anti-PEG IgM production. Such enhanced effect was abrogated by incorporation of 2'-O-methyl (2'-OMe) uridine into the sequence of siRNA, probably via inhibiting cytokine induction such as IL-6 and TNF-. Our results strongly indicate that the use of an encapsulation-type lipid nanocarrier with a low immuno-stimulatory siRNA may allow repeated dosing of siRNA containing PEGylated formulations without the induction of a strong immune reaction against PEG and thus may advance synthetic siRNA into a broad range of therapeutic applications.


Combination therapy of metronomic S-1 dosing with oxaliplatin-containing PEG-coated liposome improves antitumor activity in a murine colorectal tumor model.

Metronomic chemotherapy has been advocated recently as a novel chemotherapeutic regimen. Polyethylene glycol (PEG)-coated liposomes are well known to accumulate in solid tumors by virtue of the highly permeable angiogenic blood vessels characteristic for growing tumor tissue, the so-called "enhanced permeability and retention (EPR) effect". To expand the range of applications and investigate the clinical value of the combination strategy, the therapeutic benefit of metronomic S-1 dosing in combination with oxaliplatin (l-OHP)-containing PEG-coated liposomes was evaluated in a murine colon carcinoma-bearing mice model. S-1 is an oral fluoropyrimidine formulation and metronomic S-1 dosing is a promising alternative to infused 5-FU in colorectal cancer therapy. Therefore, the combination of S-1 with l-OHP may be an alternative to FOLFOX (infusional 5-FU/leucovorin (LV) in combination with l-OHP), which is a first-line therapeutic regimen of a colorectal carcinoma. The combination of oral metronomic S-1 dosing with intravenous administration of liposomal l-OHP formulation exerted excellent antitumor activity without severe overlapping side-effects, compared with either metronomic S-1 dosing, free l-OHP or liposomal l-OHP formulation alone or metronomic S-1 dosing plus free l-OHP. We confirmed that the synergistic antitumor effect is due to prolonged retention of l-OHP in the tumor on account of the PEG-coated liposomes, presumably via alteration of the tumor microenvironment caused by the metronomic S-1 treatment. The combination regimen proposed here may be a breakthrough in treatment of intractable solid tumors and an alternative to FOLFOX in advanced colorectal cancer therapy with acceptable tolerance and preservation of quality of life (QOL).


Evasion of the accelerated blood clearance phenomenon by coating of nanoparticles with various hydrophilic polymers.

The accelerated blood clearance (ABC) phenomenon is induced upon repeated injections of poly(ethylene glycol) (PEG)-coated colloidal carriers. It is essential to suppress this phenomenon in a clinical setting because the pharmacokinetics must be reproducible. In this study, we evaluated the induction of the ABC phenomenon using nanoparticles coated with various hydrophilic polymers instead of PEG. Nanoparticles encapsulating prostaglandin E1 were prepared by the solvent diffusion method from a blend of poly(lactic acid) (PLA) and block copolymers consisting of various hydrophilic polymers and PLA. Coating of nanoparticles with poly(N-vinyl-2-pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) led to extended residence of the nanoparticles in blood circulation in rats, although they had a shorter half-life than the PEG-coated nanoparticles. The ABC phenomenon was not induced upon repeated injection of PVP-coated nanoparticles at various time intervals, dosages, or frequencies, whereas it was elicited by PEG-coated nanoparticles. In addition, anti-PVP IgM antibody, which is estimated to be one of the crucial factors for induction of the ABC phenomenon, was not produced after injection of PVP-coated nanoparticles. These results suggest that the use of PVP, instead of PEG, as a coating material for colloidal carriers can evade the ABC phenomenon.


Optimized PEGylated adenovirus vector reduces the anti-vector humoral immune response against adenovirus and induces a therapeutic effect against metastatic lung cancer.

Application of adenovirus vectors (Adv) in metastatic cancer treatment is limited. We previously demonstrated that covalent conjugation of polyethleneglycol (PEG) to Adv enhances therapeutic effects and decreases toxic side-effects after systemic administration, but the level of immune response to PEGylated Adv (PEG-Ad) was not examined. Here, we examined the effect of PEGylation of Adv on the production of anti-Adv antibodies and antitumor response. We constructed a set of PEG-Ad using 5-kDa PEG, with modification rates of 30%, 45% and 90%. After systemic administration of Advs to rats, we examined the level of anti-Adv immunoglobulin (Ig)G and IgM in serum. The levels of anti-Adv IgG and anti-Adv IgM in rats treated with unmodified Adv were higher than those in control group. Rats treated with PEG-Ad that had a 90% modification rate showed lower level of anti-Adv IgG and anti-Adv IgM than those treated with unmodified Adv, whereas rats treated with PEG-Ad that had a 30% or 45% modification rate showed a similar level of anti-Adv IgG and IgM to those treated with unmodified Adv. Systemic administration of PEG-Ad that had a 90% modification rate, and expressed tumor necrosis factor-alpha, significantly reduced the number of metastatic colonies in the lung compared to unmodified Adv, with negligible side effects. These results suggest that systemic administration of PEG-Ad with an appropriate PEG modification rate has the potential to reduce the production of antibodies against Adv and increase the therapeutic response against metastatic cancer.


Co-administration of tacrolimus suppresses pharmacokinetic modulation of multiple subcutaneously administrated human interferon-alpha in beagle dogs.

Specific antibody production is an important issue in crossover pharmacokinetic (PK) studies of protein-based formulations. We recently reported that intravenous co-administration of tacrolimus with multiple human interferon-alpha (h-IFN) administrations successfully suppressed the production of anti-h-IFN antibodies in rats. Since crossover PK studies are preferentially carried out using larger animals such as dogs or monkeys that are capable of accepting the same dosage formulations as those for clinical use, we extended our study of co-administration of tacrolimus with multiple h-IFN administrations to beagle dogs in the present study. Beagle dogs were subcutaneously administered 0.5 million IU/kg of h-IFN once a week for 4 weeks. In some experiments, tacrolimus at 0.01 or 0.1 mg/kg was intravenously co-administered at the same time as the h-IFN administration. Co-administration of the lower dose of tacrolimus (0.01 mg/kg) failed to suppress the anti-h-IFN IgG responses, while co-administration of the higher dose (0.1 mg/kg) successfully suppressed these responses. Moreover, co-administration of tacrolimus had little effect on the serum creatinine concentrations, suggesting that multiple administrations of tacrolimus at the concentrations examined did not cause severe renal disorders. Taken together, the present data confirm that co-administration of tacrolimus is a promising way to assess crossover PK studies of human or humanized proteinic formulations in beagle dogs.


T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes.

Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [(3)H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [(3)H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.


CpG motifs in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNA-lipoplexes.

Gene therapy is largely dependent on the development of efficient delivery vehicles. To prolong their circulating time, PEGylation of the surface of a delivery vehicle is frequently applied. However, we have reported previously that anti-PEG IgM produced by intravenous injection of PEG-coated liposome is responsible for enhanced clearance of second dose PEG-coated liposomes, which is known as the "accelerated blood clearance (ABC) phenomenon." A similar phenomenon has been observed with PEG-coated pDNA-lipoplexes (PDCLs) upon their repeated injection. But the effect of the sequence of pDNA in PDCLs on inducing the ABC phenomenon has not been thoroughly investigated. Here, we focus on CpG motifs in pDNA, which are known to have a potent immune-stimulatory activity. PDCLs with non-CpG pDNA (PNDCL) diminished the anti-PEG IgM response, resulting in significant accumulation of a second dose in tumor tissue, comparable to that of a single injection, but not in enhanced accumulation in liver. In addition, PDCL induced proliferation of IgM(+) splenic cells including B cells. These results suggest that the CpG motif is a major cause of the induction of the ABC phenomenon when PDCLs are repeatedly injected. Immunogenicity is a relevant point of concern for non-viral delivery systems. Our results indicate that the use of non-CpG pDNA may allow meaningful repeated dosing of pDNA formulations without the induction of a strong immune reaction and thus may have important implications for therapeutic use of liposomal formulations of nucleic acids.


Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor.

Recently, we designed a PEG-coated cationic liposome to achieve dual targeting delivery of l-OHP to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation showed an efficient antitumor activity in a murine tumor model after three sequential liposomal l-OHP injections. This led us to assume that prior dosing with liposomes might enhance the intra-tumoral accumulation of a subsequent dose, and hence improve the therapeutic efficacy of entrapped l-OHP. The present study shows that while a single liposomal l-OHP injection does not enhance tumor accumulation of subsequent test-PEG-coated cationic liposomes, two sequential injections of liposomal l-OHP do. Cumulative cytotoxic effects of l-OHP delivered by PEG-coated cationic liposomes led to deep diffusion of a subsequent dose of liposomal l-OHP in solid tumor presumably as a result of the enlarged intra-tumoral interstitial space. Our study suggests that sequential injections of a targeted liposomal anticancer drug is of significant clinical and practical importance in enhancing the delivery of adequate quantities of anticancer agents into intractable solid tumors, and thereby may achieve a significant anticancer efficacy.


Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles.

We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).