Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models.

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Inhibitory Effects of AF-343, a Mixture of Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on Compound 48/80-Mediated Allergic Responses in RBL-2H3 Cells.

The purpose of this study was to determine the antiallergic effects of AF-343, a mixture of natural plant extracts from Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on rat basophilic leukemia (RBL-2H3) cells. The inhibitory effects on cell degranulation, proinflammatory cytokine secretion, and reactive oxygen species (ROS) production were studied in compound 48/80-treated RBL-2H3 cells. The bioactive compounds in AF-343 were also identified by HPLC-UV. AF-343 was found to effectively suppress compound 48/80-induced b-hexosaminidase release, and interleukin (IL)-4 and tumor necrosis factor-a (TNF-a) production in RBL-2H3 cells. In addition, AF-343 exhibited DPPH free radical scavenging effects in vitro (half-maximal inhibitory concentration (IC50) = 105 µg/mL) and potently inhibited compound 48/80-induced cellular ROS generation in a 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. Specifically, treatment with AF-343 exerted stronger antioxidant effects in vitro and antiallergic effects in cells than treatment with three single natural plant extracts. Furthermore, AF-343 was observed to contain bioactive compounds, including catechin, aurantio-obtusin, and chicoric acid, which have been reported to elicit antiallergic responses. This study reveals that AF-343 attenuates allergic responses via suppression of b-hexosaminidase release, IL-4 and TNF-a secretion, and ROS generation, perhaps through mechanisms related to catechin, aurantio-obtusin, and chicoric acid. The results indicate that AF-343 can be considered a treatment for various allergic diseases.

Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide.

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.

Modulation of macrophage functions by compounds isolated from Zingiber officinale.

Bioactivity-guided fractionation of Zingiber Officinale (zingiberaceae) led us to isolate 14 compounds, -gingerol ( 1), -gingerol ( 2), -gingerol ( 3), -gingerol ( 4), -paradol ( 5), -shogaol ( 6), -shogaol ( 7), 1-dehydro- -gingerdione ( 8), -gingerdione ( 9), hexahydrocurcumin ( 10), tetrahydrocurcumin ( 11), gingerenone A ( 12), 1,7-bis-(4' hydroxyl-3' methoxyphenyl)-5-methoxyhepthan-3-one ( 13), and methoxy- -gingerol ( 14). Using the RAW 264.7 cell line, the inhibitory effects on nitric oxide production induced by lipopolysaccharide and the stimulatory effects on phagocytosis of these compounds were evaluated. Compounds 7, 8, and 9 significantly decreased lipopolysaccharide-induced nitric oxide production, and compounds 7 and 8 significantly reduced inducible nitric oxide synthase expression. Among them, compound 8 also showed significant stimulatory effects on phagocytosis.

Assessment of comparative pain relief and tolerability of SKI306X compared with celecoxib in patients with rheumatoid arthritis: a 6-week, multicenter, randomized, double-blind, double-dummy, phase III, noninferiority clinical trial.

BACKGROUND: SKI306X, which consists of biologically active ingredients from Clematis mandsburica, Tricbosantbes kirilowii, and Prunella vulgaris, was developed and tested in preclinical trials in Korea. Those studies found that SKI306X was associated with an anti-inflammatory and analgesic effect, and that it can delay the destruction of cartilage in rheumatoid arthritis (RA). OBJECTIVE: The aim of this study was to compare the pain relief and tolerability of SKI306X and celecoxib in patients with RA. METHODS: This study was a 6-week, multicenter, randomized, double-blind, double-dummy, Phase III, noninferiority clinical trial. Eligible patients were aged 18 to 80 years, had a history of RA with a disease duration of > or =3 months, and were functional American College of Rheumatology (ACR) class I, II, or III before entry. After a washout period of 2 weeks, patients were randomized to SKI306X 200 mg TID or celecoxib 200 mg BID for 6 weeks. The primary end point was a change in patient assessment of pain intensity using a visual analog scale (VAS). The secondary end points were a 20% improvement in response rate as defined by the ACR (ACR20) and the frequency of rescue medication use. Results after 3 and 6 weeks of treatment were compared with baseline and between treatment groups, and all patients were assessed for adverse events (AEs), clinical laboratory data, and vital signs. AEs were identified based on spontaneous reports by patients during interviews conducted by the investigators and the study coordinator. RESULTS: Two hundred twenty-two Korean patients from 7 medical centers were assessed and 183 were enrolled and randomized to 1 of 2 treatment groups. Ninety-one patients (10 male, 81 female; mean [SD] age, 52.13 [12.64] years; mean [SD] duration of RA, 9.08 [10.23] years; no. [%] of ACR class I, II, and III, 13 [14.29], 44 [48.35] and 34 [37.36] patients, respectively) received SKI306X 200 mg TID and 92 patients (10 male, 82 female; mean [SD] age, 51.78 [10.94] years; mean [SD] duration of RA, 8.78 [7.78] years; no. [%] of ACR class I, II, and III, 14 [15.22], 44 [47.83], and 34 [36.96] patients, respectively) received celecoxib 200 mg BID. An analysis of the change in reported pain intensity as determined by VAS (mm) score between baseline and week 3 (mean [SD], 13.64 [16.62] vs 14.45 [15.89]), and between baseline and week 6 (18.4 [20.8] vs 17.9 [19.1], respectively) suggested that SKI306X was not inferior to celecoxib. The number of patients who achieved ACR20 response rate was not significantly different between the SKI306X group and the celecoxib group at week 3 (16/87 [18.4%] vs 24/87 [27.6%], respectively) and at week 6 (29/87 [33.3%] vs 29/87 [33.3%]). The frequency of rescue medication use was not significantly different between the SKI306X group and celecoxib group at week 3 (54/87 [62.1%] vs 47/87 [54.0%], respectively) or week 6 (57/87 [65.5%] vs 49/87 [56.3%]). Drug-related AEs were reported by 27 (29.7%) patients in the SKI306X group and 22 (23.9%) patients in the celecoxib group. The most frequent drug-related AEs were epigastric pain (9/91 [9.9%]) in the SKI306X group and glutamyltranferase elevation (4/92 [4.3%]) in the celecoxib group. No significant between-group differences were observed in the prevalence of drug-related clinical- or laboratory-determined AEs. CONCLUSION: The results of this study suggest that SKI306X was generally well tolerated and not inferior to celecoxib in regard to pain relief in these Korean patients with RA.

Immunosuppressive lignans isolated from Saururus chinensis.

Bioactivity-guided fractionation of Saururus chinensis (Saururaceae) using a lymphoproliferation assay led us to isolate 5 lignans (compounds 1 - 5). Compounds 1 - 5 were identified as sauchinone, (-)-saucerneol, saucerneol C, manassantin A, and manassantin B, respectively, by spectroscopic analyses. The immunosuppressive activities of the active compounds were evaluated using lymphoproliferation, mixed leukocyte response, and Th1/Th2 cytokine assays. The relative potency was in the order: manassantin A, B > (-)-saucerneol > saucerneol C > sauchinone.