Deformation characteristics of hydrogel products based on cross-linked hyaluronic acid.

BACKGROUND: The cross-linked hyaluronic acid (HA) fillers are the viscoelastic hydrogel with a dominant elasticity rather than a viscosity as a useful medical device in the soft tissue augmentation. These HA fillers undergo deformation to begin the biodegradation by the biochemical and physical environment of the body, and result of deformations are closely related to clinical performance. AIMS: The specific equation of molding index is newly generated and proved with Collin's equation, which is used for strong elastomers, for selecting optimal product in facial treatment. METHODS: In this study, the results of amplitude sweep test from five marketed HA fillers were mathematically demonstrated for the proper clinical application. RESULTS: The increment of loss modulus by deformation was evaluated as a useful factor for the maintenance of optimal shape molding performance and resistance to external deformation of the cross-linked HA gel. From this study, the equation of molding index for weak viscoelastic hydrogels like HA products can be applied for choosing which products even in aesthetic plastic field. This molding index equation is compared to Collins' equation that index of deformation as elastomer like rubber and then found the positive correlation between two equations. CONCLUSION: This study could provide a basic theory that achieve useful clinical performance according to characteristics among many types of medical devices based on the molding index.

Toxicity Assessment of a Single Dose of Poly(ethylene glycol) Diglycidyl Ether (PEGDE) Administered Subcutaneously in Mice.

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used to cross-link polymers, particularly in the pharmaceutical and biomaterial sectors. However, the subcutaneous toxicity of PEGDE has not yet been assessed. PEGDE samples (500-40,000 µg/mouse) were subcutaneously injected into the paraspinal dorsum of BALB/c male mice. Cage-side observations were carried out with measurement of organ weight, body weight variation, and feed intake, as well as histopathological characterization on day 28 post-exposure. Mice that received 40,000 µg of PEGDE showed severe toxic response and had to be euthanized. Subcutaneous injection of PEGDE did not alter feed intake and organ weight; however, the body weight variation of mice injected with 20,000 µg of PEGDE was significantly lower than that of the other groups. Exposure to 10,000 and 20,000 µg of PEGDE induced epidermal ulcer formation and hair loss. The histology of skin tissue in mice administered with 20,000 µg of PEGDE showed re-epithelialized or unhealed wounds. However, the liver, spleen, and kidneys were histologically normal. Collectively, PEGDE, particularly above 10,000 µg/mouse, caused subcutaneous toxicity with ulceration, but no toxicity in the other organs. These results may indicate the optimal concentration of subcutaneously injected PEGDE.

In vitro toxicity assessment of crosslinking agents used in hyaluronic acid dermal filler.

Hyaluronic acid (HA) dermal fillers are produced by crosslinking HA with agents, such as 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether (PEGDE) to acquire desired properties. Thus, the safety evaluation of these crosslinkers is needed at the cellular level. In the present study, cell viability, cytotoxicity, membrane integrity, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory responses were evaluated in the human keratinocyte cell line, HaCaT and human dermal fibroblast cell line, HDF in response to treatment with the crosslinkers. In both the cell lines, BDDE significantly decreased cell viability at 100-1000 ppm, while PEGDE showed a decrease at 500-1000 ppm. In HaCaT cells, BDDE markedly increased cytotoxicity (lactate dehydrogenase release) at 100-1000 ppm, but PEGDE showed an increase at 500-1000 ppm. Cells treated with BDDE (100 ppm) caused alteration in the integrity of cell membrane and shape. In both the cell lines, BDDE-treated cells showed significantly higher ROS levels and MMP loss than PEGDE-treated cells. Also, BDDE-treated cells exhibited higher COX-2 expression at 100 ppm. Expression of inflammatory cytokines (TNF-α, and IL-1 ß) was higher in BDDE-treated cells. Taken together, PEGDE-treated cells showed markedly lower cytotoxicity, ROS production, and inflammatory responses than BDDE-treated cells. Our data suggest that PEGDE is safer than BDDE as a crosslinker in HA dermal fillers.

Rheological properties and efficacy of the formulation of hyaluronic acid with tamarind seed polysaccharide for arthritis.

BACKGROUND: Tamarind seed polysaccharide (TSP) is used as a texturizing agent and a thickener in food and pharmaceutical products. There are no publications describing the addition of TSP to intra-articular injection formulations for arthritis. OBJECTIVE: The purpose of this study was to investigate the rheology and efficacy of the formulation of TSP with hyaluronic acid (HA) as a new material for injection for arthritis. METHODS: We investigated the viscoelastic properties of formulations of HA and TSP as potential lubricants for arthritis, and tested the improvement of right/left paw weight distribution in monosodium iodoacetate-induced arthritis in the rat. RESULTS: HA formulations with 3% and 4% TSP showed improved rheological characteristics and were protected against changes induced by heat sterilization. Addition of TSP also reduced pain in the arthritis model, as evidenced by normalization of the distribution of paw weight. CONCLUSIONS: TSP is a potential material as a substitute for HA or in combination with HA for intra-articular injection for arthritis.

ANTI-NOCICEPTIVE EFFECT OF AGRIMONIA EUPATORIA EXTRACT ON A CISPLATIN-INDUCED NEUROPATHIC MODEL.

BACKGROUND: Natural products including Agrimonia eupatoria are considered an incomparable source of molecular diversity that has led to the medicines, especially for pain treatment. To investigate the antinociception of Agrimonia eupatoria, we examined its activity in a rat model of cisplatin neuropathy. MATERIALS AND METHODS: Male Sprague-Dawley rats received intraperitoneal (i.p.) cisplatin twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 4 weeks. Before each injection, 2 ml of sterile saline solution was given subcutaneously to prevent renal damage via hyperhydration. The mice were treated with gabapetin as a positive control drug with a 100mg/kg intraperitoneal injection. A. eupatoria extract of 200mg/kg was solved in saline and then treated by oral administration. RESULTS: The mice treated with A. eupatoria showed lower withdrawal duration in the pin-prick and plantar tests, and a higher withdrawal threshold in the paw-withdrawal threshold test as compared to control animals in a cisplatin-induced neuropathic model. In the case of cold-allodynia, A. eupatoria treatment increased paw-withdrawal duration in a chemical test. A. eupatoria showed a more outstanding effect than gabapentin in all used tests for preventing cisplatin-induced nerve injury for 4 weeks. CONCLUSIONS: Our results suggest that A. eupatoria extract showed an antinociceptive effect in the pin-prick test, plantar test, and paw-withdrawal threshold test using a cisplatin-induced neuropathic rat model.

Antimalarial activity of nepodin isolated from Rumex crispus.

The purpose of this study is to define the antimalarial activity of Rumex crispus. To identify an active compound that is isolated from R. crispus, bioassay-based chromatographic fractionation and purification is carried out from 70 % ethanol extract of R. crispus; then, an active compound, nepodin, is identified by spectroscopic analysis. Anitmalarial activity is measured by PfNDH2 assay, cytotoxicity, and animal test. From NADH:quinone oxidoreductase enzyme (PfNDAH2) assay, nepodin exhibited significant IC50 values that were 0.74 ± 0.07 and 0.79 ± 0.06 µg/ml against P. falciparum chloroquine-sensitive (3D7) and P. falciparum chloroquine-resistant (S20), respectively. Nepodin showed a potential selective inhibition (SI index: ratio of 50 % cytotoxic concentration to 50 % effective anti-plasmodial concentration) of 161.6 and 151.4 against P. falciparum 3D7 and P. falciparum S20. In the animal test, all groups of nepodin treatment of 10, 50, and 250 mg/kg were active with a parasitemia suppression of 97.1 ± 3.3, 99.1 ± 3.7, and 99.1 ± 2.6 %, respectively. The survival time with nepodin treatment was increased by 14.6 ± 2.5, 16.2 ± 1.5, and 19.8 ± 1.7 days at each dose, respectively. This study newly identified the plant R. crispus containing nepodin, which is a potential antimalarial compound. It exhibited the inhibitory activity of PfNDH2 and prolonged the survival time on the group of nepodin treatment; moreover, it inhibited the parasitemia in the animal test.

Preclinical pharmacokinetics and biodistribution of human papillomavirus DNA vaccine delivered in human endogenous retrovirus envelope-coated baculovirus vector.

PURPOSE: Test pharmacokinetics and biodistribution of a human papillomavirus(HPV)16L1 DNA vaccine delivered in human endogenous retrovirus envelope protein (HERV)-expressing baculovirus (AcHERV) and those of naked plasmid vaccine. METHOD: HPV16L1 gene was administrated as a naked plasmid or in AcHERV to mice via intravenous and intramuscular routes. HPV16L1 gene was extracted and assayed by quantitative real-time polymerase chain reaction, which was determined to have a detection limit of 50 copies/µg genomic DNA.. RESULTS: Mean residence times of HPV16L1 in AcHERV were 4.8- and 272.2-fold higher than naked HPV16L1 DNA vaccines after intramuscular and intravenous administration, respectively. Naked HPV16L1 DNA levels 1 month after injection were >3 orders of magnitude lower in each tissue tested than AcHERV-delivered HPV16L1, which was retained in most tissues without specific tissue tropism. AcHERV-delivered HPV16L1 administered intramuscularly persisted at the injection sites. However, the levels of copy numbers in muscle were low (1,800/µg genomic DNA) after 1 month, and undetectable after 6 months. CONCLUSIONS: HPV16L1 delivered via AcHERV resides longer in the body than HPV16L1 in naked form. The lack of tissue tropism ensures the safety of AcHERV vectors for further development.

Protective effects of fucoidan against γ-radiation-induced damage of blood cells.

Fucoidan, a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and Laminaria japonica, has a variety of biological activities, including antioxidant and antitumor activities. Here, we investigated the radioprotective effects of fucoidan on human monoblastic leukemia cell line U937. Further, animal tests were carried out using Balb/c mice in order to determine the radiation-induced changes in the counts of blood cells, including thrombocytes, erythrocytes, leukocytes and hematocrit. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wherein fucoidan (1, 10, and 100 µg/mL) was observed to improve recovery from damage caused by 8-Gy radiation in a dose dependent manner. The viability of U937 cells pre-treated with fucoidan also increased in a dose dependent manner. Furthermore, fucoidan at 100 mg/kg was found to protect against changes in the counts of blood cells as follows: on day 28 after irradiation, the thrombocyte count in the irradiated controls decreased to 45% compared with the non-irradiated controls, while that in the fucoidan-treated group was 60%. The hematocrit in the fucoidan-treated group recovered to 75% on day 28, while that in the irradiated control was 68%. The erythrocyte count in the irradiated controls consistently ranged from 64% to 67% throughout the experiment, but that in the fucoidan-treated group increased gradually, ranging from 75% to 80%. The mean number of survival days and 50-day actuarial survival rate increased dose dependently in the fucoidan-treated group. The mean number of survival days and the 50-day actuarial survival rate in this group was 16, 21, and 29 days and 12%, 20%, and 30% at fucoidan doses of 1, 10, and 100 mg/kg. The values of these parameters in the control group were 9 days and 0%, although the difference between the test and control groups was not statistically significant. Our results may prove valuable in the field of radioprotection.

Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii.

The Actinomycete strain KH29 is antagonistic to the multidrug-resistant Acinetobacter baumannii. Based on the diaminopimelic acid (DAP) type, and the morphological and physiological characteristics observed through the use of scanning electron microscopy (SEM), KH29 was confirmed as belonging to the genus Streptomyces. By way of its noted 16S rDNA nucleotide sequences, KH29 was found to have a relationship with Streptomyces cinnamonensis. The production of an antibiotic from this strain was found to be most favorable when cultured with glucose, polypeptone, and yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic produced was identified, through comparisons with reported spectral data including MS and NMR as a cyclo(L-tryptophanyl-L-tryptophanyl). Cyclo(L-Trp-L-Trp), from the PY cultures of KH29, was seen to be highly effective against 41 of 49 multidrugresistant Acinetobacter baumannii. Furthermore, cyclo(LTrp- L-Trp) had antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Saccharomyces cerevisiae, Aspergillus niger, and Candida albicans, However, it was ineffective against Streptomyces murinus.

Pre- and co-treatment with xanthophyll enhances the anti-leukemic activity of adriamycin.

Xanthophyll (lutein) is one of the most potent known antioxidants. It has been shown that dietary intake of xanthophyll helps to prevent age-related macular degeneration and the development of cataracts. It may also reduce the risk of developing various types of cancer. Here we showed that xanthophyll efficiently scavenged the stable free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) with an IC50 of 0.5mM and effectively countered the cytotoxic effect of tert-butylhydroperoxide (tBuOOH) on various leukemic cell lines. In contrast, oxidized xanthophyll did not have these effects. We then examined whether dietary intake of xanthophyll inhibited leukemic tumor growth in mice injected subcutaneously with the leukemic cell line L1210. After one month, treatment with 13mg/kg xanthophyll had inhibited tumor growth by about 20%. Xanthophyll also enhanced the anti-leukemic activity of adriamycin in the L1210 mouse model as it extended the duration of adriamycin-induced suppression of tumor growth. Moreover, the two agents together reduced tumor volume by about 50% whereas treatment with adriamycin alone only stalled growth for a few days. Oxidized xanthophyll did not have any anti-leukemic effects on its own or in combination with adriamycin. Thus, the radical scavenging activity of the food supplement xanthophyll prevents oxidative stress, inhibits leukemic tumor growth, and enhances the anti-leukemic activities of a common chemotherapeutic agent in a synergistic manner.

Radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lung.

In the present study, we investigated the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lungs to determine its potential as a radioprotective agent. We found that early lung damage induced by irradiation was reduced by treatment with 40 mg/kg of cyclo(Lphenylalanyl- L-prolyl) in the latent and early pneumonitis phases. Expression of TNF-alpha and TGF-beta1 at 2 and TGF-beta1 at 8 weeks post-irradiation was decreased in animals that received both radiation and cyclo(L-phenylalanyl-L-prolyl) compared with animals that received radiation alone. Evidence indicated that the proinflammatory cytokine TNF-alpha and the fibrogenic cytokine TGF-beta1 likely play a role in the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl). However, besides TNF-alpha and TGF-beta1 expressions, the precise mechanism by which cyclo(L-phenylalanyl-L-prolyl) ameliorates the induced radiation damage is not clear.

Glycoprotein isolated from Acanthopanax senticosus protects against hepatotoxicity induced by acute and chronic alcohol treatment.

The protective effect of a 30 kDa glycoprotein (GF-AS) isolated from the stem bark of Acanthopanax senticosus against acute and chronic alcohol-induced hepatotoxicity were studied. N-terminal amino acid sequence of GF-AS showed NH(2)-Val-Ala-Tyr-Pro-Trp-Ala-Gly-Phe-Ala-Leu-Ser-Leu-Glx-Pro-Pro-Ala-Gly-Tyr-. GF-AS significantly increases the activities of alcohol-metabolizing enzymes, including alcohol dehydrogenase, microsomal ethanol metabolizing system, and acetaldehyde dehydrogenase in rats acutely treated with alcohol, resulting in decreased plasma alcohol levels. GF-AS also increases the activities of antioxidant enzymes and glutathione level. Markers of liver injury induced by alcohol: elevated serum levels of aspartate aminotransferase, alanine aminotransferase, triglyceride and cholesterol, are reduced by GF-AS in both acutely and chronically treated rats. The activities of lipogenic enzymes including malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphoglucuronic acid dehydrogenase in chronic alcohol-treated rats are significantly decreased by GF-AS. Furthemore, GF-AS improves histological change in fatty liver and hepatic lesions induced by alcohol. Collectively, GF-AS may alleviate alcohol-induced hepatotoxicity through increasing ethanol and lipid metabolism, as well as antioxidant defense systems in livers injured by acute- and chronic-alcohol treatment.

Anti-angiogenic effect of the seed extract of Benincasa hispida Cogniaux.

Benincasa hispida in Korea was used mainly diabetes and diuresis diseases. This study was carried out to evaluate anti-angiogenic effect of the seed extract of Benincasa hispida Cogniaux. Basic fibroblast growth factor (bFGF) is a potent angiogenic factor found in various tumors. In this study, we found that the seed extract of Benincasa hispida Cogniaux decreased bFGF-induced endothelial cell proliferation and tube formation in a dose-dependent manner. Besides, Benincasa hispida seed extract showed no cytotoxicity on HUVECs and normal fibroblast cells. Furthermore, the seed extract of Benincasa hispida showed a potent inhibitory effect on bFGF-induced angiogenesis in vivo. These results suggest that the seed extract of Benincasa hispida inhibits the proliferation of endothelial cells induced by bFGF, which may explain its anti-angiogenic properties.

Correlative effect between in vivo hollow fiber assay and xenografts assay in drug screening.

PURPOSE: This study was carried out to assess the usage of an in vivo hollow fiber assay to screen drugs with highly predictive accuracy. MATERIALS AND METHODS: The assay systems used were the hollow fiber and xenografts assays. The hollow fiber assay was carried out with the following steps; preparation of fibers, preparation of cells, loading and implanting fibers, treatment with drugs, removal of fibers and assaying for the cell viability by the MTT assay. For the xenografts assay, cell suspensions were subcutaneously transplanted into the mice. Therapy was started when the tumor volume reached 100 approximately 200 mm(3). The tumor volumes were calculated using the formula V=[length+(width)(2)]/2, and used for evaluating the efficacy of the drugs. The drug treatment doses used were adriamycin 2.1 mg/kg, mitomycin-C 0.25 mg/kg, 5-fluorouracil 24.5 mg/kg and paclitaxel 2.5 mg/kg, and administrated intravenously five times daily. RESULTS: The correlation between the xenografts and hollow fiber assays was evaluated in 20 tumor cell lines and 4 anti-cancer agents. In the 20 tumor cell lines, the overall predictive accuracy of the hollow fiber assay for sensitivity was 83%, with a predictive accuracy for resistance of 92%. CONCLUSION: The hollow fiber assay was assessed as effective in drug efficacy evaluation, and found to be compatible with that of the xenografts assay.

Anti-metastatic activity of glycoprotein fractionated from Acanthopanax senticosus, involvement of NK-cell and macrophage activation.

Previously, we reported that water-extracted Acanthopanax senticosus exhibited anti-metastatic activity by stimulating the immune system. In this study, we fractionated glycoproteins (EN-SP) from the soluble protein layer (GF-AS) of A. senticosus and determined their basic chemical properties. We also investigated the anti-tumor and immunostimulating activities of the fractionated glycoprotein, EN-SP. We found that intravenous (i.v.) administration of GF-AS dramatically inhibited metastasis of colon26-M3.1 carcinoma cells to the lung in a dose-dependent manner. In vitro analysis showed GF-AS to enhance the proliferation of splenocytes. GF-AS also stimulated peritoneal macrophage, which was followed by the production of various cytokines such as IL-1beta, TNF-alpha, IL-12 and IFN-gamma. Furthermore, the production of these cytokines was partially blocked when peritoneal macrophage was cultured with the polyclonal antibodies against GF-AS. The depletion of NK cells by rabbit anti-asialo GM1 serum partly abolished the inhibitory effect of GF-AS on lung metastasis of colon26-M3.1 cells. Using gel filtration, EN-SP, an active glycoprotein fraction, is isolated from GF-AS. While both GF-AS and EN-SP stimulated the proliferatation of splenocytes of normal mice, EN-SP showed higher anti-metastatic activity and more potently stimulated the proliferation of splenocytes compared to GF-AS. These results suggest the use of EN-SP, the fractionated glycoprotein from A. senticosus, can be used as a therapeutical reagent to prevent or inhibit tumor metastasis.

Synthesis and biological activity of the new 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid.

A series of 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid 6, were synthesized and evaluated for their antitumor activity.