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Comprehensive analyses of intracellular disposition and

We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (∼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.

Carcinoembryonic antigen (CEA) is a tumor-specific antigen overexpressed in multiple cancers. CEA is expressed as a membrane protein, a part of which is cleaved from the cell membrane and secreted into blood. The soluble form of CEA (sCEA) has been shown to accelerate the clearance of anti-CEA antibody, which limits the antibody distribution in the tumor. To overcome this issue, we developed an anti-CEA monoclonal antibody, 15-1-32, which shows a strong affinity for membrane-bound CEA (mCEA) and relatively weak affinity for sCEA. In this study, we compared the effect of sCEA on the pharmacokinetics of 15-1-32 in mice with that of another anti-CEA monoclonal antibody, labetuzumab, showing less selectivity to mCEA than 15-1-32. As expected, the effect of sCEA on the serum concentration of 15-1-32 was much smaller than that of labetuzumab. The decrease in the area under the curve (AUC) of serum concentration was 22.5% for 15-1-32 when it was coadministered with sCEA, while that of labetuzumab was 79.9%. We also compared the pharmacokinetics of these two antibodies in CEA-positive tumor-bearing mice. The AUCs of 15-1-32 and labetuzumab were decreased in tumor-bearing mice compared with non-tumor-bearing mice to a similar extent (approximately 40% decrease). These results suggested that mCEA also contributes to the clearance of anti-CEA antibodies in CEA-positive tumor-bearing mice. Although the increased selectivity to mCEA minimized the effect of sCEA on the pharmacokinetics of 15-1-32, it may be insufficient to improve the pharmacokinetics in CEA-positive cancer patients. SIGNIFICANCE STATEMENT: Because previous studies reported the rapid clearance of anti-CEA antibodies mediated by soluble CEA, we obtained a monoclonal antibody, 15-1-32, selective to membrane-bound CEA and evaluated the effects of CEA on its pharmacokinetics. Although the effect of soluble CEA on the serum concentration of 15-1-32 was very small, the clearance of 15-1-32 in CEA-positive tumor-bearing mice was still rapid, suggesting membrane-bound CEA also contributes to the clearance of anti-CEA antibodies. These results indicated that increasing selectivity to membrane-bound CEA is not enough to improve the pharmacokinetics of anti-CEA antibody.

In vivo biodistribution analyses, especially in tumors, of nucleic acids delivered with nanoparticles are important to develop drug delivery technologies for medical use. We previously developed wrapsome® (WS), an ~100 nm liposomal nanoparticle that can encapsulate siRNA, and reported that WS accumulates in tumors in vivo and inhibits their growth by an enhanced permeability and retention effect. In the present study, we evaluated the pharmacokinetics of nucleic acid-containing nanoparticles by combining dynamic positron emission tomography (PET) imaging and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. An 18-mer phosphorothioate oligodeoxynucleotide (ODN), trabedersen, was used as a model drug and was encapsulated in WS. Dynamic PET imaging and time-activity curve analysis of WS-encapsulated 64Cu-labeled ODNs administered to mice with MIA PaCa-2 subcutaneous xenograft tumors showed tumor accumulation (~3% injected dose per gram (%ID/g)) and liver accumulation (~30 %ID/g) at 24 h. Under these conditions, LC/MS/MS analysis showed that the level of intact ODNs was 1.62 %ID/g in the tumor and 1.70 %ID/g in the liver. From these pharmacokinetic data, the intact/accumulated ODN ratios were calculated using the following equation: intact/accumulated ODN ratio (%) = %ID/g LC/MS/MS, tissue, mean/%ID/g PET, tissue, mean × 100. Interestingly, the ratios for the tumor and kidney were maintained at 20–50% over 48 h after administration of the WS-encapsulated form. In contrast, the ratio for the liver rapidly decreased at 24 h, showing the same pattern as that for naked ODN. These different patterns indicate that WS effectively protected the ODN in the tumor and kidney, but protected it less efficiently in the liver. A combined approach of dynamic PET imaging and LC/MS/MS analysis will assist the development of nanoparticle-encapsulated nucleic acid drugs, such as those using WSs, to determine their detailed pharmacokinetics.

Photodynamic therapy (PDT) utilize a photosensitizing agent and light for cancer therapy. It exerts anti-cancer effect mainly by inducing vascular occlusion at the irradiated site. By controlling the irradiation area, PDT can be used in a tumor-specific manner. However, the non-specific cellular damage in the surrounding normal tissue is still a serious concern. Photoimmunotherapy (PIT) is a new type of targeted cancer therapy that uses an antibody-photon absorber conjugate (APC). The superiority of PIT to PDT is the improved target specificity, thereby reducing the damage to normal tissues. Here, we developed a novel APC targeting epithelial cell adhesion molecule (EpCAM) as well as a negative control APC that does not bind to the EpCAM antigen. Our in vitro analysis of APC cytotoxicity demonstrated that the EpCAM APC, but not the negative control, was cytotoxic to EpCAM expressing COLO 205 cells after photoirradiation, suggesting that the cytotoxicity is antigen-dependent. However, in our in vivo analysis using a mouse xenograft tumor model, decreased volume of the tumors was observed in all the mice treated with irradiation, regardless of whether they were treated with the EpCAM APC or the negative control. Detailed investigation of the mechanism of these in vivo reveal that both APCs induce vascular occlusion at the irradiation site. Furthermore, the level of vascular occlusion was correlated with the blood concentration of APC, not the tumor concentration. These results imply that, similar to PDT, PIT can also induce non-targeted vascular occlusion and further optimization is required before widespread clinical use.

Carcinoembryonic antigen (CEA) is a classic tumor‐specific antigen that is overexpressed in several cancers, including gastric cancer. Although some anti‐CEA antibodies have been tested, to the best of our knowledge, there are currently no clinically approved anti‐CEA antibody therapies. Because of this, we have created the novel anti‐CEA antibody, 15‐1‐32, which exhibits stronger binding to membrane‐bound CEA on cancer cells than existing anti‐CEA antibodies. 15‐1‐32 also shows poor affinity for soluble CEA; thus, the binding activity of 15‐1‐32 to membrane‐bound CEA is not influenced by soluble CEA. In addition, we constructed a 15‐1‐32‐monomethyl auristatin E conjugate (15‐1‐32‐vcMMAE) to improve the therapeutic efficacy of 15‐1‐32. 15‐1‐32‐vcMMAE showed enhanced antitumor activity against gastric cancer cell lines. Unlike with existing anti‐CEA antibody therapies, antitumor activity of 15‐1‐32‐vcMMAE was retained in the presence of high concentrations of soluble CEA.

Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.

薬物の多くは，血漿中でアルブミンやα1-酸性糖タンパクなどのタンパク質へ結合している．アルブミンは脂溶性の高い酸性化合物，α1-酸性糖タンパクは塩基性化合物に対し高い親和性を示す．タンパクへ結合した薬物は細胞膜を透過することができないため，血漿中の遊離型薬物のみが組織に分布して薬効や毒性を発現し，代謝や排泄を受けて体内から除去される．したがって，血漿中タンパク結合は薬物の体内動態や薬効，毒性に多大な影響を及ぼす．血漿中タンパク結合には種差があり，体内動態や薬効，毒性の種差の原因となる．また，血漿中タンパク結合は病態や薬物間相互作用によっても変動し，薬効の減弱や副作用の増強など臨床上好ましくない現象を引き起こす場合がある．したがって，薬物の血漿中タンパク結合は医薬品の探索・開発を通じて評価しなければならない重要な項目の一つである．

The role of breast cancer resistance protein (BCRP/ABCG2) in limiting the brain and testis penetration of xenobiotic compounds in the blood-brain and -testis barriers was investigated using Bcrp(-/-) mice. Tissue/plasma concentration ratios in the brain (K(p,brain)) and testis (K(p,testis)) obtained under steady-state conditions were significantly increased in Bcrp(-/-) mice for PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), N-hydroxyl PhIP, MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), dantrolene, and prazosin. In addition, the K(p,brain) of triamterene and the K(p,testis) of 4'-hydroxyl PhIP were also significantly increased in Bcrp(-/-) mice. The effect of functional impairment of Bcrp on the brain uptake of PhIP, dantrolene, and daidzein in Bcrp(-/-) mice determined using in situ brain perfusion was weaker than that observed on the K(p) values. In vitro transcellular transport experiments using cell lines expressing mouse Bcrp or P-glycoprotein (Mdr1a/Abcb1a) showed that, among the tested Bcrp substrates, PhIP, MeIQx, prazosin, and triamterene are common substrates of Bcrp and P-glycoprotein. The K(p) values of common substrates exhibited a smaller increase both in the brain and testis of Bcrp(-/-) mice than expected from the in vitro Bcrp activities. The Bcrp-specific substrates were weak acids, whereas basic or neutral BCRP substrates were also P-glycoprotein substrates. These results suggest that BCRP limits the tissue penetration of xenobiotic compounds in the blood-brain and -testis barriers, but its in vivo importance is also modulated by P-glycoprotein activity.

Troglitazone sulfate (TGZS) is the major metabolite of troglitazone (TGZ), an antidiabetic agent, and thought to be a cause of the cholestasis induced by TGZ. The aim of the present study is to elucidate the involvement of breast cancer resistance protein (BCRP/ABCG2) in the hepatic disposition of TGZS. The basal-to-apical transport of TGZS was enhanced in organic anion transporting polypeptide 1B1-expressing Madin-Darby canine kidney II cells by infection of recombinant adenovirus harboring human BCRP and mouse Bcrp cDNA. TGZS was given to wild-type and Bcrp (-/-) mice by constant infusion. Biliary excretion is the predominant elimination pathway of TGZS in wild-type mice, and the biliary excretion clearance of TGZS with regard to the hepatic concentration was reduced to 30% of the control in Bcrp (-/-) mice. However, plasma and hepatic concentrations were unchanged, suggesting induction of compensatory mechanisms in Bcrp (-/-) mice for the elimination of TGZS. Involvement of BCRP in the intestinal efflux transport of TGZS was examined using everted sacs. The mucosal efflux clearance of TGZS showed only a slight reduction (15% reduction) in Bcrp (-/-) mice. Our results suggest that BCRP plays a major role in the biliary excretion but a minor role in the intestinal transport of TGZS.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance.

Background: Heparin-binding EGF-like Growth Factor (HB-EGF), a member of the EGF family, exerts its biological activity through activation of the EGF receptors. HB-EGF is initially synthesized as a membrane-anchored precursor protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF plays pivotal roles in many physiologic and pathologic processes such as development and cell proliferation. In this study, we measured soluble HB-EGF concentrations in serum samples obtained from healthy volunteers and patients of various cancer types. Materials and methods: Soluble HB-EGF levels in human serum samples were quantified by the immuno-PCR method. Results: The mean soluble HB-EGF levels of the 20 healthy volunteers and 10 colon, breast, ovarian, head and neck, non-small cell lung, pancreatic, and small cell lung cancer patients were 5.04, 18.0, 15.1, 10.4, 6.69, 9.78, 23.6, and 5.60 pg/mL, respectively. There was a statistically significant difference between the HB-EGF concentrations of healthy volunteers and patients in 5 out of 7 cancer types. Furthermore, a trend for HB-EGF levels to increase along with disease stage was observed. Conclusion: Soluble HB-EGF may be a useful diagnostic serological biomarker for various cancer types, and a predictive and/or pharmacodynamic biomarker for HB-EGF-targeted therapeutics.

The effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of the phytoestrogens daidzein, genistein, and coumestrol was investigated using Bcrp(-/-) mice. Expression of the genes for either mouse Bcrp or human BCRP in MDCK II cells induced apically directed transport of the three phytoestrogens, whereas their transcellular transport was identical in mock and LLC-PK1 cells expressing mouse Mdr1a. After oral administration, the plasma levels of daidzein and genistein were increased in Bcrp(-/-) mice, but only a minimal change was observed for coumestrol. At steady state, tissue-to-plasma concentration ratios of the three phytoestrogens in the brain and testis of wild-type mice were very small and similar to those of [(14)C]inulin, whereas those were significantly increased in the brain and testis of Bcrp(-/-) mice. The largest increases were observed with genistein (9.2- and 5.8-fold in the brain and testis, respectively). The distributions of genistein in the epididymis and fetus, but not the ovary, were also increased in Bcrp(-/-) mice. The Bcrp protein was localized in the luminal membrane of the endothelial cells in the testis and the body of the epididymis and in both the luminal and abluminal side of ducts in the head of the epididymis. These results suggest that Bcrp limits the oral availability and distribution into the brain and testis, epididymis, and fetus of phytoestrogens.

Breast cancer resistance protein (Bcrp/Abcg2) is a member of the ATP-binding cassette transporter family with the ability to transport a variety of sulfate conjugates. In the present study, the regional expression and activity of Bcrp and sulfotransferases (SULTs/Sults) were investigated in mouse intestine. Western blotting analysis revealed the highest expression of Bcrp in the ileum over the duodenum, jejunum, and colon. Functional analysis of Bcrp was performed in everted intestinal sacs using 4-methylumbelliferone (4MU). The mucosal secretion clearance of 4MU sulfate formed in the enterocytes was markedly reduced in the jejunum, ileum, and colon of Bcrp (-/-) mice in comparison with wild-type mice, whereas a slight and nonsignificant reduction was observed in the duodenum. The reduction in the mucosal secretion clearance was most marked in the ileum followed by the colon and jejunum. In addition, the mucosal secretion clearance of minoxidil sulfate, an active metabolite of minoxidil, was also significantly reduced in the intestine of Bcrp (-/-) mice. The sulfation activity of 4MU was higher in the colon than in the small intestine where glucuronidation activity was somewhat higher than the sulfation activity. Real-time polymerase chain reaction analysis showed that the expression of sulfotransferases, such as Sult1a1/2, Sult1b1, and Sult1d1, was also highest in the colon. These results suggest that Bcrp activity is higher in the mid to lower intestine and that the cooperation of Bcrp and SULT provides an important detoxification pathway, particularly in the colon.

The synthesis of a series of 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methyl-2-arylbenzofuran and 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methylbenzofuran-2-carboxamide derivatives as novel alpha(2C)-adrenergic receptor antagonists are described. Their affinity at three different human alpha(2)-adrenergic receptors is reported, and some of these compounds exhibited high affinity for the alpha(2C)-adrenergic receptor with high subtype selectivity. Among them, compound 10e has been found to show the anti-L-dopa-induced dyskinetic activity in marmosets. The structure-activity relationship of these compounds is also discussed.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.