Takao Taki

Anti-neovascular therapy using novel peptides homing to angiogenic vessels.

Cancer chemotherapy targeted to angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels formed by a dorsal air sac method from a phage-displayed peptide library. Three distinct phage clones that markedly accumulated in murine tumor xenografts presented PRPGAPLAGSWPGTS-, DRWRPALPVVLFPLH- or ASSSYPLIHWRPWAR-peptide respectively. After the determination of the epitope sequences of these peptides, we modified liposomes with epitope penta-peptides. Liposome modified with APRPG-peptide showed high accumulation in murine tumor xenografts, and APRPG-modified liposome encapsulating adriamycin effectively suppressed experimental tumor growth. Finally, specific binding of APRPG-modified liposome to human umbilical endothelial cells, and that of PRP-containing peptide to angiogenic vessels in human tumors, i.e., islet cell tumor and glioblastoma, were demonstrated. The present study indicates the usefulness of APRPG-peptide as a tool for anti-neovascular therapy, a novel modality of cancer treatment.

Antibody to Ga1NAc-GD1a and Ga1NAc-GM1b in Guillain—Barré syndrome subsequent to Campylobacter jejuni enteritis

N-Acetylgalactosaminyl GD1a (GalNAc-GD1a) is a proposed target molecule for serum antibody in some patients with Guillain-Barré syndrome (GBS) (Kusunoki et al., 1994). We examined autoantibody to GalNAc-GD1a in sera from 58 GBS patients. Eight GBS patients had high IgG anti-GalNAc-GD1a antibody titers, 3 of whom also had high IgM anti-GalNAc-GD1a antibody titers. These 8 patients had experienced gastrointestinal infection before the onset of their neurological symptoms. Campylobacter jejuni was isolated from 4 of them. An absorption test indicated the presence of the GalNAc-GD1a epitope in lipopolysaccharides of C. jejuni. Sera that had anti-GalNAc-GD1a antibody reacted with several acidic glycolipids in bovine peripheral nerve, one of which was identified as N-acetylgalactosaminyl GM1b (GalNAc-GM1b). Serum binding to GalNAc-GM1b was decreased by absorption with GalNAc-GD1a. The presence of GalNAc-GM1b as well as GalNAc-GD1a has been reported in human peripheral nerves. We assume that C. jejuni, which bears the [GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-3 GalNAc beta 1-] epitope, is the immunogen and that the glycoconjugates with the epitope are target molecules for the autoantibody in peripheral nerves of some GBS patients.

Sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy

Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.

Anti-neovascular therapy by liposomal drug targeted to membrane type-1 matrix metalloproteinase

Because membrane type‐1 matrix metalloproteinase (MT1‐MMP) is expressed specifically on the angiogenic endothelium as well as tumor cells, an agent possessing the ability to bind to this molecule might be useful as a tool for active targeting of tumor angiogenic vessels. Based on the sequences of peptide substrates of MT1‐MMP, which had been determined by using a phage‐displayed peptide library, we examined the binding ability of peptide‐modified liposomes for endothelial cells and targeting ability for tumor tissues by positron emission tomography (PET). Liposomes modified with stearoyl‐Gly‐Pro‐Leu‐Pro‐Leu‐Arg (GPLPLR‐Lip) showed high binding ability to human umbilical vein endothelial cells and accumulated in the tumor about 4‐fold more than did the unmodified liposomes. Because we reported previously that liposomalized 5′‐O‐dipalmitoylphosphatidyl 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (DPP‐CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, strongly suppressed tumor growth when delivered in liposomes modified with another angiogenic homing peptide, we examined the antitumor activity of DPP‐CNDAC entrapped in GPLPLR‐Lip. DPP‐CNDAC/GPLPLR‐Lip showed significant tumor growth suppression compared to DPP‐CNDAC/unmodified liposomes. These results suggest that DPP‐CNDAC‐liposomes modified with MT1‐MMP‐targeted peptide are useful for cancer anti‐neovascular therapy (ANET), namely, tumor growth suppression by damage to angiogenic endothelial cells.

Immunosuppressive constituents from Saussurea medusa.

The methanol extract of Saussurea medusa Maxim afforded two lignans: 2alpha-guaicyl-4-oxo-6alpha-catechyl-3,7-dioxabicyclo [3.3.0]octane and 1alpha-hydroxy-2alpha,4alpha-guaicyl-3,7-dioxabicyclo[3.3.0]octane; two chlorophyll derivatives: 13-epi-phaeophorbide-a and 13-epi-phaeophorbide-a methyl ester; one megastigmane derivative: 3beta-hydroxy-5alpha,6alpha-epoxy-7-megastigmen-9-one, along with 19 known compounds. Their structures were established on the basis of spectroscopic studies.

Immunosuppressive terpenoids from extracts of Tripterygium wilfordii

Abstract The clinically used extract (TΠ) of Tripterygium wilfordii Hook f. give 19 new compounds, including five kaurane diterpenes (1–5), one manoyl oxide diterpene (6), and one abietane diterpene (7), three ursene triterpenes (8, 9 and 15), six oleanane triterpenes (10–13, 16 and 19), and three friedelane triterpenes (14, 17 and 18), as well as 15 known compounds (20–34). Their structures were elucidated by spectroscopy and X-ray analysis. Based on the screening of isolated compounds and other compounds reported in previous papers [J. Nat. Prod. 62 (1999) 1522; J. Nat. Prod. (2001) in press; Phytochemistry 53 (2000) 805], we identified the main components that are responsible for the therapeutic effect of TΠ.

Anti-neovascular therapy by liposomal DPP-CNDAC targeted to angiogenic vessels

We previously reported that liposomalized 5′‐O‐dipalmitoylphosphatidyl 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (DPP‐CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti‐neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage‐displayed random peptide library, and observed that peptide‐modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP‐CNDAC‐liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG‐modified DPP‐CNDAC‐liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP‐CNDAC‐liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG‐liposomes and non‐modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG‐modified DPP‐CNDAC‐liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti‐neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.

Subclass of IgG antibody to GM1 epitope-bearing lipopolysaccharide of Campylobacter jejuni in patients with Guillain-Barré syndrome

Sera of patients who develop Guillain-Barré syndrome (GBS) subsequent to Campylobacter jejuni enteritis frequently have IgG anti-GM1 antibody. Lipopolysaccharide (LPS) of C. jejuni isolated from a GBS patient has a GM1 ganglioside-like structure. IgG subclass distribution of the anti-GM1 antibody in GBS patients is mainly restricted to IgG1 and IgG3. Since IgG antibodies to bacterial polysaccharide generally are restricted to IgG2 subclass, some investigators have assumed that either the general rules for immune response to LPS are broken in the patients or an alternative antigen has yet to be identified. To clarify whether the LPS participates in the production of the anti-GM1 antibody, we investigated the subclass of IgG antibody to the LPS that bears GM1-like structure. The subclasses of IgG antibody to the LPS were restricted predominantly to IgG1 and IgG3. The GM1 epitope-bearing LPS may function in the production of the anti-GM1 antibody in patients with GBS subsequent to C. jejuni infection.

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).

Ganglioside-like epitopes of lipopolysaccharides from Campylobacter jejuni (PEN 19) in three isolates from patients with Guillain-Barré syndrome

Sera from patients with Guillain-Barré syndrome (GBS) frequently have anti-GM1 antibody. We earlier showed that an lipopolysaccharides (LPS) from Campylobacter jejuni (PEN 19) isolated from a GBS patient has a GM1 ganglioside-like structure. Aspinall et al. (Biochemistry, 61 (1994) 335-337) reported that OH 4382 has an LPS that bears a CD3 ganglioside-like structure and that OH 4384 has an LPS that bears a GT1a-like structure; both strains were isolated from patients with GBS. They also suggested a GM1-like structure is present in the LPSs from OH 4384, but failed to show the presence in the LPSs from OH 4382. To clarify the pathogenesis of GBS after infection by C. jejuni (PEN 19), we investigated the carbohydrate structures of the three strains by thin-layer chromatography immunostaining with cholera toxin and monoclonal anti-ganglioside antibodies. We found that both OH 4382 and OH 4384 have an LPS with the GM1 epitope as well as one with the GT1a or GD3 epitope.