Semisynthesis and antibacterial activities of nidulin derivatives.

Derivatives of the fungal depsidone, nidulin, have been synthesized in order to evaluate the potential of the chemical skeleton as antibacterial agents. Alkylation, acylation, and arylation reactions of nornidulin underwent in a regioselective manner to predominantly produce 8-O-substituted derivatives. Many of the semisynthetic derivatives showed more potent antibacterial activities than nidulin, In particular, 8-O-aryl ether derivatives displayed significant activities against Gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus.

Potential anticancer activity of lichen secondary metabolite physodic acid.

Secondary metabolites present in lichens, which comprise aliphatic, cycloaliphatic, aromatic and terpenic compounds, are unique with respect to those of higher plants and show interesting biological and pharmacological activities. However, only a few of these compounds, have been assessed for their effectiveness against various in vitro cancer models. In the present study, we investigated the cytotoxicity of three lichen secondary metabolites (atranorin, gyrophoric acid and physodic acid) on A375 melanoma cancer cell line. The tested compounds arise from different lichen species collected in different areas of Continental and Antarctic Chile. The obtained results confirm the major efficiency of depsidones. In fact, depsides atranorin and gyrophoric acid, showed a lower activity inhibiting the melanoma cancer cells only at more high concentrations. Whereas the depsidone physodic acid, showed a dose-response relationship in the range of 6.25-50 µM concentrations in A375 cells, activating an apoptotic process, that probably involves the reduction of Hsp70 expression. Although the molecular mechanism, by which apoptosis is induced by physodic acid remains unclear, and of course further studies are needed, the results here reported confirm the promising biological properties of depsidone compounds, and may offer a further impulse to the development of analogues with more powerful efficiency against melanoma cells.

In vitro antitumor activities of the lichen compounds olivetoric, physodic and psoromic acid in rat neuron and glioblastoma cells.

Context Since methods utilised in the treatment of glioblastoma multiforme (GBM) are inadequate and have too many side effects, usage of herbal products in the treatment process comes into prominence. Lichens are symbiotic organisms used for medicinal purposes for many years. There are various anticancer treatments about components of two lichen species used in the present study. Objective Antitumor potential of three lichen secondary metabolites including olivetoric acid (OLA) and physodic acid (PHA) isolated from Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) and psoromic acid (PSA) isolated from Rhizoplaca melanophthalma (DC.) Leuckert (Lecanoraceae) were investigated on human U87MG-GBM cell lines and primary rat cerebral cortex (PRCC) cells for the first time. Materials and methods PRCC cells used as healthy brain cells were obtained from Sprague-Dawley rats. The treatments were carried out on the cells cultured for 48 h. Cytotoxic effects of different concentrations (2.5, 5, 10, 20 and 40 mg/L) of metabolites on the cells were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) analyses. Total antioxidant capacity (TAC) and total oxidant status (TOS) parameters were used for assessing oxidative alterations. Oxidative DNA damage potentials of metabolites were investigated via evaluating 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels. Results Median inhibitory concentration (IC50) values of OLA, PHA and PSA were 125.71, 698.19 and 79.40 mg/L for PRCC cells and 17.55, 410.72 and 56.22 mg/L for U87MG cells, respectively. It was revealed that cytotoxic effects of these metabolites showed positive correlation with concentration, LDH activity and oxidative DNA damage. Discussion and conclusion The present findings obtained in this study revealed that primarily OLA and then PSA had high potential for use in the treatment of GBM.

In vitro effects of preserved and unpreserved anti-allergic drugs on human corneal epithelial cells.

PURPOSE: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. METHODS: Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. RESULTS: The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. CONCLUSIONS: The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures.

[Toxicity of topical ocular anti-allergic agents on human corneal epithelial cells in vitro].

OBJECTIVE: To investigate the cytotoxic effect of three kinds of topical ocular anti-allergic agent, including olopatadine 0.1% (A group), ketotifen fumarate 0.025% (B group) and pemirolast potassium 0.1% (C group), on cultured human corneal epithelial cells in vitro. METHODS: Primary human corneal epithelial cells were cultured with keratinocyte serum-free medium. The cells were exposed to three kinds of topical ocular anti-allergic agent for a period of 10 min, 30 min, 2 h, 6 h, 12 h and 24 h. Toxicity was examined in three ways. MTT assay was used to quantify cytotoxicity. Cell membrane permeability and intracellular esterase activity were analyzed with live-dead viability staining of fluorescent calcein-AM/ethidium homodimer. The morphologic analysis was performed by light and scanning electron microscopy. Statistical methods adopted one-way ANOVA (analysis of variance) and Student-Newman-Keuls q test between each group. The P-value of 0.05 was considered statistically significant. RESULTS: (1) Morphologic changes: The Findings under the light microscopy were demonstrated that cells became round or edematic and detached from dishes after exposure to topical ocular anti-allergic agent. The cellular damage was more severe with longer exposure time and increasing concentration. Likewise, the electron microscopy examination showed reduced microvilli with longer exposure time and increasing concentration. The cellular changes of 20.0% olopatadine 0.1% were reduced when compared to the other agents. (2) Live/dead viability/cytotoxicity assay: Ethidium homodimeric permeates damaged cell membranes and results in red fluorescence. These results indicated that cell membrane damage caused by 20.0% olopatadine 0.1% at 6, 12, 24 h was less than those of ketotifen fumarate 0.025% and pemirolast potassium 0.1%. The data of A group were (29.7 +/- 2.6)%, (36.9 +/- 3.2)%, (51.2 +/- 4.3)%, B group were (36.5 +/- 3.1)%, (48.5 +/- 4.3)%, (75.5 +/- 3.8)% and C group were (37.1 +/- 2.2)%, (52.7 +/- 3.4)%, (71.1 +/- 5.1)%, respectively. The q values of A to B group and A to C group at 6 h were 3.27, 4.31, respectively (P < 0.05). The green fluorescent staining of calcein-AM indicated intracellular esterase activity was decreased after incubation with increasing concentration and longer exposure time. There was no significantly different result between each group (P > 0.05). The proportion of green staining cell of A, B and C group at 24 h were 100.0% with 50.0% concentration and were (23.2 +/- 4.6)%, (29.5 +/- 5.2)%, (31.1 +/- 5.5)% respectively with 20.0% concentration (F = 1.97, P = 0.377). (3) MTT assay: The results of the three groups revealed cell viability decreased significantly with increasing concentration and longer exposure time at all the concentrations except 0.8%. MTT values for A, B and C group at the concentration of 20.0%, at 6 h were 0.429 +/- 0.028, 0.367 +/- 0.038, 0.379 +/- 0.012 and 4% at 24 h were 0.457 +/- 0.025, 0.401 +/- 0.008, 0.387 +/- 0.012, respectively. The data for olopatadine 0.1% were significantly improved over those of ketotifen fumarate 0.025% and pemirolast potassium 0.1%. The q value of A to B group, A to C group were 3.01, 3.77 (P < 0.05) at the concentration of 20.0%, 6 h and were 3.63, 4.11 (P < 0.05) at the concentration, 24 h. There were no statistical significant results at other concentrations. CONCLUSIONS: The topical ocular anti-allergic agent, olopatadine 0.1%, showed less toxic effects on human corneal epithelial cells compared to ketotifen fumarate 0.025% and pemirolast potassium 0.1%. Olopatadine 0.1% may offer a safer option to the corneal epithelium when used to treat allergic keratoconjunctivitis over an extended period of time.

Comparison of the conjunctival toxicity of topical ocular antiallergic agents.

PURPOSE: The aim of this study was to investigate the cytotoxic effect of topical ocular allergic agents with H1-receptor antagonism and inhibition of histamine release from mast cells on the cultured conjunctival cells of rabbit in vitro. METHODS: Cell damage by the topical ocular antiallergic agents (azelastine hydrochloride, ketotifen fumarate, and olopatadine hydrochloride) was determined by using the lactate dehydrogenase (LDH) leakage assay with the rate of dilution of 10, 20, and 30%, respectively, for a period of 0 and 30 min and 4, 12, and 24 h, and compared with the balanced salt solution-treated group. The osmolarity, pH, composition of electrolytes, preservatives, and morphologic findings of all the antiallergic agents were also evaluated. RESULTS: The LDH titers increased after all the antiallergic agents were exposed up to 4 h, maintained its level for 12 h, and then decreased until 24 h. There was no statistical significance among the three agents. The greater titer of LDH, the more conjunctival cells became swollen or round. Azelastine and ketotifen showed greater LDH titer, edema, and cytoplasmic and nuclear degenerations of the conjunctival cells than that of olopatadine. The levels of Na(+), Cl(-), and pH were significantly lower with azelastine and ketotifen, compared with olopatadine, and all antiallergic agents contained the same concentration of benzalconium chloride. CONCLUSIONS: When antiallergic agents are used to treat allergic conjunctivitis other than olopatadine, a particularly toxic effect on conjunctival cells associated with azelastine and ketotifen, rather than olopatadine, should be considered clinically.

Prolongation of antipyretic action and reduction of gastric ulcerogenicity in the rat by controlled-release granules of bermoprofen, a new nonsteroidal anti-inflammatory drug.

While bermoprofen [(+-)-10,11-dihydro-alpha,8-dimethyl-11- oxodibenz[b,f]oxepin-2-acetic acid], a new nonsteroidal antiinflammatory drug (NSAID), has potent antipyretic and analgesic activities with a short biological half-life, it shows ulcerogenic activity as a side-effect like other nonsteroidal anti-inflammatory drugs. A bermoprofen preparation was specially designed to prolong its duration of action and to reduce its side effect. Immediate-release granules (IRGs) were prepared by coating particles of Nonpareil 103 with bermoprofen. The IRGs were also coated by spraying a film solution composed of ethylcellulose and hydroxypropylmethylcellulose (3:2), and, thereby, three kinds of retard-release granules (RRGs) were obtained with the coating amounts of 1.5, 2.5, and 4%. Peak plasma bermoprofen levels were seen 0.5, 1, 2, and 3 h after single oral administrations of IRG, RRG(1.5%), RRG (2.5%), and RRG(4%), respectively, in rats. The potency order in ulcerogenic activity was RRG(1.5%) greater than IRG greater than RRG(2.5%) greater than RRG(4%) after a single oral dosage in rats. Then, IRG and RRG(4%) were mixed (in the ratio 3:7, which was calculated from their simulated plasma bermoprofen profiles) in order to get fast onset and long duration of antipyretic action. These mixed granules [i.e., the sustained-release granules (SRGs)] exhibited a falt plasma bermoprofen profile, and longer lasting antipyretic and lower ulcerogenic activities in rats in comparison with IRG or bermoprofen. From these results, it is suggested that a bermoprofen SRG preparation has more durable therapeutic activity and lower side-effects as a NSAID.

A postulated mechanism of beta-sympathomimetic induction of rib and limb anomalies in rat fetuses.

Treatment of gravid rats (days 6-15 of gestation) with the beta-sympathomimetic doxaminol resulted in wavy ribs and bent limbs in the offspring. The fetuses also exhibited defective mineralization. These anomalies were produced by pharmacologically effective doses of the drug. Prior treatment with the beta-receptor blocker carazolol prevented their formation, so that the beta-sympathomimetic action of doxaminol is evidently a causative factor. Various hypotensive agents whose activity is not mediated by beta-receptors failed to produce abnormalities. This eliminates the possibility of a non-specific etiology such as diminished placental perfusion. The cyclooxygenase inhibitor indomethacin lowered the incidence of wavy ribs. Furosemide, a loop diuretic that stimulates renal prostaglandin synthesis, increased the incidence of abnormalities when combined with doxaminol. The nature of the anomalies found suggests that 1) fetal compression by the myometrium and 2) defective mineralization are prerequisites for their development. The first condition could be produced via the complex mechanism of beta-sympathomimetic-induced stimulation of prostaglandin synthesis. Defective mineralization can result directly from cAMP-mediated activation of osteoclasts and possibly be further promoted by beta-sympathomimetic-mediated prostaglandin action on the osteoclast. The pathological findings in the fetal rat skeleton cannot be correlated with corresponding findings in human neonates whose mothers were subjected to prolonged therapeutic uterine relaxation with beta 2-sympathomimetics, for example. Since the anomalies in the rat disappear spontaneously in the post-natal period, their clinical relevance appears to be slight.

Mutagenicity of lichen constituents.

Usnic acid (the most abundant lichen constituents), physodic, and physodalic acids isolated from Hypogymnia enteromorph (Ach.) Nyl. were tested for mutagenicity in the Ames Salmonella/microsome assay. Physodalic acid exhibited clear dose-related mutagenicity against Salmonella typhimurium strain TA 100 with or without S9 mix in both plate-incorporation and preincubation assays. The addition of S9 mix increased the number of revertants approximately threefold and fourfold in preincubation and plate-incorporation assays, respectively.

The pharmacological profile of 2-(8-methyl-10,11-dihydro-11-oxodibenz[b,f]oxepin-2-yl)propionic acid (AD-1590), a new non-steroidal anti-inflammatory agent with potent antipyretic activity.

Anti-inflammatory, analgesic, antipyretic and gastrointestinal ulcerogenic activities of 2-(8-methyl-10,11-dihydro-11-oxodibenz(b,f]oxepin-2-yl)propionic acid (AD-1590), a new non-steroidal anti-inflammatory drug, were compared with indomethacin (INN: indomethacin) and other non-steroidal anti-inflammatory drugs (NSAID) in experimental animals. AD-1590 showed the potent inhibitory activity on acute and subacute inflammation such as carrageenin hind paw edema (oral ED50 = 1.35 mg/kg), acetic acid-induced increased vascular permeability (0.205 mg/kg), UV-erythema (0.295 mg/kg) and felt pellet-induced granuloma formation (1.7 mg/kg), and its potency was on the whole 2 to 3 times that of indomethacin. Oral analgesic ED50-values of AD-1590 were 0.245, 8.32 and 13.9 mg/kg in the writhing tests, and 2.45 mg/kg in the silver nitrate-induced arthritic pain test. Analgesic potency of AD-1590 was on the whole comparable to that of indomethacin. Against the pyrexia caused by two kinds of pyrogens (yeast and adjuvant), AD-1590 exerted a strong antipyretic action at oral doses as low as 0.02 to 0.1 mg/kg, and its potency (ED50 equal 0.0210 and 0.0406 mg/kg) was 8.7 to 11 times that of indomethacin. , AD-1590 displayed the antipyretic activity at low doses which were widely different from its anti-inflammatory and analgesic effective dose. The body temperature was not affected by 20 mg/kg p.o. of AD-1590 in the afebrile animals. AD-1590 was the strongest antipyretic drug among 10 NSAID tested. In rats, AD-1590 produced gastrointestinal ulcer similar to indomethacin, and its gastric ulcerogenicity (SUD50 equal 13.8 mg/kg p.o.) was about one-half that of indomethacin. The activity of AD-1590 in the fecal occult bleeding test in beagle dogs was weaker than that of indomethacin. The potency of AD-1590 (IC50 equal 0.78 mumol/l) as a prostaglandin synthetase inhibitor was about 2.7 times that of indomethacin in the in vitro test. The safety index (SUD50/ED50) of AD-1590 was larger than that of indomethacin, extremely large (the index equal 657 and 340) in the antipyretic activity. Besides, acute lethal toxicity of AD-1590 (oral LD50 equal 147 mg/kg in rats, 500 mg/kg in mice) was about 1/8 and 1/24 that of indomethacin. From these results, it was suggested that AD-1590 had extraordinarily potent antipyretic activity, potent anti-inflammatory activity superior to indomethacin, analgesic activity equivalent to indomethacin, and a wide safety margin.

Nonsteroidal antiinflammatory agents. 2. Derivatives/analogues of dibenz[b,e]oxepin-3-acetic acid.

6,11-Dhydro-11-oxodibenz[b,e]oxepins and some related compounds have been synthesized and evaluated for antiinflammatory effect according to the carrageenan paw edema method in rats. The structure-activity relationships have been discussed among acetic acid, carboxylic acid, alcohol, and tetrazole derivatives of dibenzoxepins and acetic acid derivatives of thienobenzoxepins and of the corresponding thiepins. The 3-isopropyl alcohol 9 and 11-deoxo-3-propionic acid (49) were more active than indomethacin but not as active as the title compound (i.e., 43). Carboxylic acids, tetrazoles, esters, amides, and ketones were less active than the corresponding acetic acids. Three compounds (31, 33, and 34) were evaluated for ulcerogenicity and lethality but none surpassed 6,11-dihydro-11-oxodibenz[b,e]oxepin-3-acetic acid (41) in therapeutic ratio.

Acute, subacute and chronic toxicity of oxepinac.

The acute, subacute and chronic toxicity of 6,11-dihydro-11-oxodibenz[b,e]oxepin-3-acetic acid (oxepinac) was investigated in several animal species. The LD50 value was lower in rats than in rabbits, mice and dogs. The major cause of death was perforative ulcer in the gastrointestinal tract. Long-term study in rats revealed that oxepinac produced no hematological, blood chemical and pathological changes except for minor anemia and fatal ulcer formation occurring predominantly in females. Oxepinac proved to be less toxic than indometacin in chronic toxicity in rats.

Pharmacology of a new non-steroidal, anti-inflammatory agent: 6,11-dihydro-11-oxodibenz [b, e] oxepin-2-acetic acid (HP 549).

HP 549 is an orally effective non-steroidal anti-inflammatory agent with moderate analgesic and antipyretic activity. It is active in adjuvant-induced polyarthritis when given prophylactically or therapeutically. HP 549 also inhibits carrageenan-induced paw edema in the rat, an activity which is not altered by adrenalectomy. The analgesic activity of HP 549 was demonstrated in phenylquinone writhing. However, HP 549 produced variable results in the Randall-Selitto analgesia test. The anti-pyretic activity of HP 549 appears to be weak. HP 549, unlike other pharmacologically active anti-inflammatory drugs, does not produce gastric irritation at effective doses and is 45 times less ulcerogenic than indomethacin. Also the acute therapeutic indices for HP 549 are more favorable than for indomethacin.