Structural Fingerprints of an Intact Monoclonal Antibody Acquired under Formulated Storage Conditions via 15N Direct Detection Nuclear Magnetic Resonance.

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15N direct detection nuclear magnetic resonance (NMR) technique, the 15N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15N direct detection.

Karataviosides G-K, five new bisdesmosidic steroidal glycosides from the bulbs of Allium karataviense.

We have analyzed the steroidal glycosides in Allium karataviense bulbs, and isolated five new bisdesmosidic steroidal glycosides: karataviosides G-K (1-5). The structures were elucidated by extensive spectroscopic analysis, including 2D NMR and enzymatic and hydrolytic cleavage. Karatavioside G (1) is an entirely novel furostanol glycoside, which has an O-ß-d-glucopyranosyl-(1→6)-ß-d-glucopyranosyl-(1→6)-ß-d-glucopyranosyl unit at C-26 of the aglycone. Although a variety of cholestanol glycosides have been isolated, mainly from Liliaceae and Agavaceae, karataviosides J and K (4 and 5) are also notable because they are the most polar cholestanol bisdesmosides discovered, in which a lycotetraose is attached to C-3 of the aglycone, and a glucose or O-glucosyl-(1→3)-glucose is attached at C-16. The isolated glycosides were also evaluated for their cytotoxic activities against cultured tumor cell lines.

Optimizing the Physicochemical Properties of Raf/MEK Inhibitors by Nitrogen Scanning.

Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1 (CH5126766/RO5126766) was selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors.

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC50=8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED50=4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment.

Fluorine Scanning by Nonselective Fluorination: Enhancing Raf/MEK Inhibition while Keeping Physicochemical Properties.

A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives, iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.

Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog.

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.

Ornithosaponins A-D, four new polyoxygenated steroidal glycosides from the bulbs of Ornithogalumthyrsoides.

By analyzing the steroidal glycoside content of fresh bulbs of Ornithogalum thyrsoides (Liliaceae), we were able to isolate four new polyoxygenated steroidal glycosides, which we named ornithosaponins A-D (1-4). The structures of 1-4 were elucidated on the basis of extensive spectroscopic analysis, including that of 2D NMR data, and the results of acidic or alkaline hydrolysis. The aglycone structure of 1-4 has not been previously reported. It is also notable that ornithosaponins B-D (2-4) have been found to contain 6-deoxy-beta-D-gulopyranose as a sugar component, which is rarely encountered in plant glycosides.

27-norlanostane glycosides from the bulbs of Muscari paradoxum.

Eight 27-norlanostane glycosides (1-8), including five new compounds (3 and 5-8), were isolated from the MeOH extract of the bulbs of Muscari paradoxum. The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage. The cytotoxic activity of 1-8 against HSC-2 human oral squamous cell carcinoma cells is also reported.

Steroidal glycosides from the bulbs of Ornithogalum thyrsoides.

Phytochemical analyses have been carried out on the fresh bulbs of Ornithogalum thyrsoides with particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol saponins and seven new cholestane glycosides, together with three known steroidal compounds. The structures of the new glycosides were determined on the basis of their spectroscopic data, including 2D NMR spectroscopy, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells.

[Anti-allergic action effect of Pseudolarix amabilis Rehd. extract and its efficacy on atopic dermatitis].

Pseudolarix amabilis Rehd. extract was examined in vitro for antibacterial effects, anti-inflammatory effects, and inhibitory effects on histamine release. Pseudolarix amabilis Rehd. extract was also examined for efficacy on dermatitis in atopic dermatitis model mice (NC mice) and effects on keratinous moisture level and transepidermal water loss in miniature pigs. Pseudolarix amabilis Rehd. extract had antibacterial effects on Staphylococcus aureus, Candida albicans, and Streptococcus pyogenes; however this antibacterial effect varied with the temperature at which and conditions under which Pseudolarix amabilis Rehd. was extracted. Pseudolarix amabilis Rehd. extract at the final concentration of 2 mg/mL significantly inhibited the hyaluronidase activity; and at 0.005, 0.05, and 0.5 mg/mL, it also significantly inhibited the histamine release. In the mice in which atopic dermatitis had been induced, 28-day administration of Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly inhibited aggravation of dermatitis without having effects on body weight. In the dorsal skin of miniature pigs, Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly increased keratinous moisture level with the increase in the number of dosing days, and caused no changes in transepidermal water loss. From the above results, it is clear that Pseudolarix amabilis Rehd. extract inhibits both proliferation of bacteria and inflammation caused by antigens. Furthermore, it is suggested that Pseudolarix amabilis Rehd. extract will serve as a medicinal drug which effectively moistens the skin and prevents and heals dermatitis.

A novel evaluation method of gastric mucoadhesive property in vitro and the mucoadhesive mechanism of tetracycline-sucralfate acidic complex for eradication of Helicobacter pylori.

PURPOSE: The gastric mucoadhesive property of tetracycline-sucralfate acidic complex (CO) was evaluated by using a novel method in vitro to compare with the in vivo test. The mucoadhesive mechanism of the acidic complex was also studied. METHODS: The gastric mucosa removed from a rat was placed covering the end of a plunger and secured in a disposable syringe. The acidic test medium was gradually infused in and then flowed out. Two different kinds of CO, tetracycline, or a physical mixture (PM) were introduced into the device to compare their mucoadhesive properties. The tetracycline content in the residue on the mucosa was measured. The results were compared with those of the in vivo test. The acidic response of CO and the protein binding capacity of a sucrose octasulfate group (SOS) in sucralfate or CO were evaluated. RESULTS: The mucoadhesive properties of CO were clearly superior to those of PM. The remaining amounts of tetracycline in each test sample, determined by the in vitro test, were in agreement with those of the in vivo test. The excellent mucoadhesive property of CO appeared to be caused by the rapid response to the acid and resulting mucoadhesive gel formation. Furthermore, the binding capacity of SOS to the protein was clearly greater than that of PM. The excessive acid treatment during the preparation of CO tended to decrease the mucoadhesive property. CONCLUSIONS: CO appeared to be potentially useful for the eradication of Helicobacter pylori because of the direct delivery of tetracycline to the gastric mucosa for an extended period of time.

[Analysis of scratching behavior in a picryl chloride induced atopic dermatitis model in mice].

To elucidate the actual state of scratching behavior of NC mice noted when PiCl-induced dermatitis occurs, the circadian rhythm in scratching behavior of this mouse model was examined, and the time when scratching behavior, which is useful to evaluate the severity of itch, occurs was assessed. A steroid drug (Prednisolone ointment), which has been confirmed to inhibit dermatitis from worsening, was used to examine whether or not, or how it inhibits scratching behavior in this mouse model. It became clear that scratching behavior increased during a period from the evening to the night in the animals which had not been sensitized (normal animals); compared with the day time, scratching behavior occurred more often in the nighttime. It also became clear that scratching behavior increased in the animals with PiCl-induced dermatitis increase in the frequency of induction of dermatitis, and Prednisolone ointment significantly inhibited scratching behavior in the animals in which dermatitis had been induced with PiCl six times. From these results, it can be said that scratching behavior increases in PiCl-induced mouse atopic dermatitis models correlatively with the increase in the frequency of induction of dermatitis, and steroid drugs decrease the frequency of the scratching behavior. In conclusion, it is strongly suggested that this mouse model is useful for development of therapeutic methods and novel medicinal drugs for atopic dermatitis.

Lanosterol and tetranorlanosterol glycosides from the bulbs of Muscari paradoxum.

Three tetranorlanostane glycosides, named lucilianosides C-E, and three lanostane glycosides, named scillasaponins E-G, together with a known lanostane glycoside, were isolated from the MeOH extract of Muscari paradoxum (Liliaceae) bulbs, as confirmed by detailed analysis of their 1H, 13C, and two-dimensional NMR spectroscopic data, and by the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activity against HSC-2 human oral squamous cell carcinoma cells.

Norlanostane and lanostane glycosides from the bulbs of Chionodoxa luciliae and their cytotoxic activity.

Ten lanostane glycosides (1-10), including two new norlanostane glycosides (2 and 7) and a new lanostane glycoside with a spirolactone ring system (9), were isolated from the fresh bulbs of Chionodoxa luciliae (Liliaceae). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis and the results of hydrolytic cleavage to be (23S)-3beta-[(O-beta-D-apiofuranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->3)]-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl)oxy]-17alpha,23-epoxy-28,29-dihydroxy-27-norlanost-8-en-24-one (2), (23S)-17alpha,23-epoxy-29-hydroxy-3beta-[(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->3)]-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl)oxy]-27-norlanost-8-ene-15,24-dione (7), and (23S,25R)-17alpha,23-epoxy-29-hydroxy-3beta-[(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->3)]-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl)oxy]lanost-8-en-23,26-olide (9), respectively. The cytotoxic activity of the isolated compounds against HSC-2 human oral squamous cell carcinoma cells are also reported.