Thermo-sensitive gel in glaucoma therapy for enhanced bioavailability: In vitro characterization, in vivo pharmacokinetics and pharmacodynamics study.

AIMS: Glaucoma is a chronic ophthalmic disease, which has become one of the leading causes to progressive and irreversible blindness. Current ophthalmic drug delivery to treat glaucoma is mostly eyedrop, whose rapid elimination on corneal surface can lead to poor bioavailability. The present study was aimed to develop a timolol maleate loaded thermo-sensitive gel (TM-TSG) with improved bioavailability to treat glaucoma. MAIN METHODS: TM-TSG was prepared by homogeneously dispersing 0.3% (w/v) timolol maleate, 24.25% (w/v) poloxamer 407 (P407) and 1.56% (w/v) poloxamer 188 (P188) into phosphate buffer solution (pH = 7.4) and the formulated TM-TSG was characterized. KEY FINDINGS: TM-TSG was stored in liquid form at room temperature (25 °C) and transited to semisolid gel at physiological temperature (32 °C). The rheological property of TM-TSG was in favor of uniform distribution of drug. TM-TSG showed good stability at different conditions including centrifugation, autoclaving and different temperature. In vivo pharmacokinetic studies indicated that TM-TSG could enhance absorption of TM in aqueous humor and improve the ocular bioavailability in comparison of commercial TM eyedrops. In vivo experiment result showed that TM-TSG had greater effect in treating glaucoma than TM eyedrops by sustainably lowering intraocular pressure (IOP) for a week. Moreover, slit lamp test and histopathological analysis demonstrated that TM-TSG had excellent biocompatibility. SIGNIFICANCE: TM-TSG could be a promising ophthalmic delivery system for glaucoma therapy.

Rikkunshito induces gastric relaxation via the ß-adrenergic pathway in Suncus murinus.

BACKGROUND: Rikkunshito (RKT) is a gastroprotective herbal medicine. In this study, we investigated the role of RKT in the relaxation of the gastric body (fundus and corpus) and antrum. METHODS: We used Suncus murinus, a unique small model animal with similar gastrointestinal motility to humans and dogs. RKT was added at 0.1, 1.0, and 5.0 mg/mL to induce relaxation in vitro; the outcome measure was the intensity of relaxation. The number of spontaneous antral contractions in the absence or the presence of RKT was also counted. KEY RESULTS: Rikkunshito induced the relaxation of the gastric body and antrum and decreased the number of spontaneous antral contractions in a dose-dependent manner. The responses to RKT (1.0 mg/mL) were not affected by pretreatment with atropine, N-nitro-l-arginine methyl ester, ritanserin, or ondansetron. On the other hand, timolol almost completely reversed the relaxation induced by RKT (1.0 mg/mL) on the gastric body and antrum and the occurrence of the spontaneous antral contractions. Both butoxamine, a ß(2) -adrenoreceptor antagonist, and L 748337, a ß(3) -adrenoreceptor antagonist, but not CGP 20712, a ß(1) -adrenoreceptor antagonist, significantly reversed the RKT-induced (1.0 mg/mL) gastric relaxation. CONCLUSIONS & INFERENCES: These results indicate that RKT stimulates and modulates gastric relaxation through ß(2) - and ß(3) -adrenergic, but not ß(1) -adrenergic, pathways in S. murinus.

Activity of crude cold-water extract of the culinary-medicinal oyster mushroom, Pleurotus ostreatus (Jacq.:Fr.) P.Kumm. (higher Basidiomycetes), and timolol maleate on induced ocular hypertension.

Aqueous crude cold-water extract from the fruiting body of the culinary-medicinal oyster mushroom Pleurotus ostreatus was assessed for activity against increased intra-ocular pressure (IOP) in mice. A 0.1% dexamethasone instillation was used to raise the intra-ocular pressure in the animals above the 21-mmHg threshold limit. The extract has intrinsic anti-hypertensive properties that are dose dependent. A comparison analysis indicated that 150 mg/mL of the crude extract produced 57.69% reduction in the intra-ocular pressure, while doses of 100 mg/ mL and 200 mg/mL produced 44.78% and 70.03% IOP reduction, respectively, compared with timolol maleate with 57.69%. The results were significant at 0.05 confidence limit (p < 0.05) when compared to a placebo and therefore support its use for the treatment of increased intra-ocular pressure.

Efficacy of tetrandrine on lowering intraocular pressure in animal model with ocular hypertension.

PURPOSE: To compare the efficacy of topical tetrandrine (an alkaloid isolated from the Chinese medicinal herb Radix Stephania tetrandrae S) and timolol 0.5% ophthalmic solution on lowering the intraocular pressure (IOP) in ocular normotensive and hypertensive rats. METHODS: The experiment was designed as 2 parts. In the first part, normal male Sprague-Dawley rats were divided into 4 groups followed by topical administration once of 0.1%, 0.2%, 0.3% tetrandrine, and 0.5% timolol in the right eye, 0.9% saline was administered once at the opposite eye as control. In the second part, the ocular hypertension model was induced unilaterally in the rats by a diode laser treatment. Twice daily applications of the above drugs were delivered to hypertensive eyes. The control group was administered 0.9% saline. The TonoPen XL tonometer was used to determine the IOP levels. RESULTS: No lowering effect on IOP was detected in the normotensive rats treated with tetrandrine whereas timolol significantly reduced IOP in normotensive eyes. Both tetrandrine and timolol significantly reduced the IOP levels in the hypertensive eyes compared with the levels in the saline-treated groups. All concentrations of tetrandrine used in this study showed significant reduction of IOP in the laser-induced hypertensive eyes. Tetrandrine 0.3% had a similar efficacy as 0.5% timolol in reducing elevated IOP in ocular hypertensive eyes. CONCLUSIONS: This study provides evidence that tetrandrine has a major effect on lowering IOP levels in the ocular hypertension rat model. The functional mechanisms of tetrandrine require further investigation.

Mechanisms underlying mechanical responses to Ephedra herb of isolated rabbit urinary bladder and urethra, a possible stress urinary incontinence therapeutic.

To compare the mechanisms underlying mechanical responses to ephedrine and Ephedra herb, a main component of Kakkon-to, in isolated male and female rabbit urinary bladder and urethral strips, responses of isolated strips to the agents were recorded in organ bath systems. Ephedrine and Ephedra herb relaxed the female urinary bladder to the similar extent. These relaxations are reversed to contractions by timolol. In the presence of timolol, ephedrine produced less contraction of urethral strips in the female than those in the male; this contraction was abolished by prazosin. Ephedra herb contracted the female urethra less than that of the male, and the contraction was stronger than that by ephedrine. The contraction caused by Ephedra herb in strips treated with timolol was significantly inhibited by prazosin. The prazosin-resistant contraction of the female urethra was greater than that of the male. Quinacrine, a phospholipase A(2) inhibitor, indomethacin, and AA861, a 5-lipoxygenase inhibitor, inhibited the contraction. The contraction was inhibited by ZK 158252, a leukotriene (LT) B(4)-receptor antagonist. These findings suggest that Ephedra herb contracts the urethra via arachidonic acid metabolites together with alpha(1)-adrenoceptor stimulation. The metabolites produced by 5-lipoxygenase may stimulate LTB(4), but not CysLt(1), receptors. These contractile components induced by Ephedra herb and Kakkon-to might be effective for the treatment of stress urinary incontinence.

Comparative efficacy of pilocarpine, timolol and latanoprost in experimental models of glaucoma.

Intraocular pressure (IOP)-lowering effects of investigational antiglaucoma drugs often need comparison with existing drugs, but detailed data showing comparative efficacy of antiglaucoma drugs with different mechanism of action has not been reported so far. This study was designed to establish baseline information of the IOP-lowering effect of three currently used antiglaucoma drugs in three experimental models in rabbits, so that they act as a benchmark for the efficacy evaluation of the future experimental antiglaucoma drugs. The IOP-lowering effect of single-drop application of pilocarpine, timolol and latanoprost was studied in normotensive, water loading and steroid-induced models of glaucoma in rabbits. The noncontact tonometer was used for the first time to estimate IOP in rabbits. The peak IOP-lowering effect of pilocarpine, timolol and latanoprost in normotensive rabbit eye was 18.23%, 20% and 22.56%, respectively. In water-loading model, the maximum protection against the rise in IOP was shown by latanoprost (40.27%), followed by timolol (31.39%) and pilocarpine (28.91%). In steroid-pretreated rabbit eyes, peak IOP-lowering effects of pilocarpine, timolol and latanoprost were 25.65%, 34.21% and 35.06%, respectively. Therefore, the latanoprost was found to be most effective in all three models followed by timolol and pilocarpine. The results of this study can be used for future preclinical investigations for the assessment of IOP-lowering activity of potential antiglaucoma drugs.

Human lens epithelial cell damage and stimulation of their secretion of chemical mediators by benzalkonium chloride rather than latanoprost and timolol.

OBJECTIVE: To investigate the effects of latanoprost, timolol maleate, and benzalkonium chloride on cell damage and induction of the secretion of chemical mediators of stress and wound healing by human lens epithelial cells in culture. METHODS: Cells from a human lens epithelial cell line (SRA01/04) were cultured in Dulbecco minimum essential medium supplemented with 5% fetal bovine serum. The amounts of latanoprost (50 micro g/mL), timolol maleate (5 mg/mL), or benzalkonium chloride (200 micro g/mL) used in eyedrops, and x10 to x1000 dilutions thereof, were added to the medium. After 7 days' culture, cell morphological changes were assessed using phase-contrast microscopy, and cell-free culture supernatants were collected for prostaglandin E2 (PGE2), interleukin 1alpha (IL-1alpha), and interleukin 6 (IL-6) iodine I 125 radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent enzyme immunoassay, respectively. RESULTS: All cells that were cultured with the concentrations of latanoprost, timolol, or benzalkonium chloride used in eyedrops detached from the culture dish and died within 3 days. At a x10 dilution of latanoprost or timolol or a x100 dilution of benzalkonium chloride, no proliferation or elongation of the cells was observed. Secretions of PGE2, IL-1alpha, and IL-6 at x10 dilutions of latanoprost or timolol were 3 to 77 times higher than in controls, whereas they were 190 to 305 times higher at a x180 dilution of benzalkonium chloride. The amounts of these soluble mediators in culture supernatants depended on the dose of latanoprost, timolol, or benzalkonium chloride added. CONCLUSION: Our results indicate that benzalkonium chloride, used as the preservative in eyedrops containing latanoprost or timolol, is the agent most damaging to lens epithelial cells and most strongly stimulates the expression of soluble chemical mediators in these cells.

Tracheal relaxant activity of cissaglaberrimine and trilobinine, two aporphinic alkaloids from Cissampelos glaberrima.

In the present work we studied the relaxant effect of two aporphinic alkaloids isolated from the roots barks of Cissampelos glaberrima St. Hil. (Menispermaceae), (+)-cissaglaberrimine (CGE; a tertiary alkaloid) and (+)-trilobinine (TBE; a quaternary alkaloid). In guinea-pig tracheal preparations, CGE and TBE reduced the spontaneous tone and inhibited the contractions induced by carbachol and histamine. TBE was 6 times more potent than CGE in reducing the spontaneous tone and it was also 1.5 times more potent in antagonising the effects of carbachol and histamine. The inhibitory effect of CGE in contractions induced by histamine was not attenuated in the presence of timolol (10 microM). However, in the same experimental conditions, timolol almost completely abolished the inhibitory effect of TBE. These results showed that the relaxations induced by TBE were dependent on the activation of beta 2-adrenoceptors, while the mechanism involved in relaxations induced by CGE remains to be determined.

Dark adaptation in early primary biliary cirrhosis.

BACKGROUND: The role of vitamin A in early primary biliary cirrhosis (PBC) remains uncertain. METHODS: We assessed dark adaptation and assayed vitamin-A-related compounds in 10 patients with early PBC and a group of age- and sex-matched controls. RESULTS: In patients compared with controls: (i) mean final light threshold value was 11.8% greater (p < 0.004), (ii) time taken to see the first light stimulus was longer (2.8 +/- 0.6 vs 1.4 +/- 0.2 min, mean +/- SEM; p < 0.03) and (iii) sensitivity to light stimuli was impaired after 6 min in the dark (p < 0.03). Three patients had an abnormal final light threshold despite receiving regular vitamin A; two had a low serum vitamin A. Raised serum bilirubin and increased age were the most important determinants of impaired dark adaptation. CONCLUSIONS: Patients with early PBC have modestly impaired dark adaptation, despite standard vitamin A supplementation, although these changes may not have a significant effect on visual function. Vitamin A supplementation should be recommended for older patients with jaundice, but its effect should be carefully monitored.

The preclinical pharmacology of dorzolamide hydrochloride, a topical carbonic anhydrase inhibitor.

Dorzolamide hydrochloride (S,S-5,6-dihydro-4H-4-ethylamino-6-methylthieno [2,3-b]thiopyran-2-sulfonamide-7,7-dioxide HCl; MK-507; L-671,152) is a water-soluble, potent inhibitor of human carbonic anhydrase isoenzymes II and IV in vitro, the respective IC50 values being 0.18 nM and 6.9 nM. In contrast, it was found to be a much weaker inhibitor of human carbonic anhydrase isoenzyme I (IC50 value of 600 nM). The topical administration of one 50 microliters drop of 0.5%, 1% and 2% solutions of dorzolamide maximally lowered the intraocular pressure (IOP) of glaucomatous monkeys by 22%, 30% and 37%, respectively. Good ocular hypotensive activity was also observed in ocular normotensive and hypertensive rabbits. Its site of action was within the eye, and the reductions in IOP in both species was achieved via decreased aqueous humor inflow. The duration of action of 2% dorzolamide was shorter than that of 0.5% timolol in glaucomatous monkeys. The IOP lowering activity of timolol in this paradigm was enhanced by the concomitant instillation of dorzolamide. Both acutely and repeatedly administered 2% dorzolamide did not decrease regional ocular blood flow in the rabbit, and the topical instillation of the drug had no adverse effects on the eye of rabbits, dogs and monkeys. Dorzolamide has been approved in a number of countries for use in patients with ocular hypertension or open-angle glaucoma.

Effect of dietary polyunsaturated fatty acids on contraction and relaxation of rat femoral resistance arteries.

We investigated the effects of dietary polyunsaturated fatty acids (PUFA) derived from fish oil (n-3 PUFA) and plant seed oil (n-6 PUFA), in amounts relevant to human consumption, on the alpha 1-adrenoceptor-mediated contractile responses of isolated rat resistance arteries. Rats were fed semisynthetic diets, deriving 40% of total calories from fat. The control diet, which had sufficient linoleic acid to prevent essential fatty acid deficiency, had a polyunsaturated/saturated fatty acid (P/S) ratio of 0.3. The n-3 PUFA were given as a daily oral supplement of fish oil. For the n-6 PUFA diet, the proportion of linoleic acid in the diet was increased to obtain P/S ratio of 2.0. Diets were administered for 8 weeks. At the end of the feeding period, second-order branches of the femoral artery (< 300-micros diameter) were mounted in pairs in an isometric myograph, and responses to norepinephrine (NE) 3 nM-10 microM with addition of yohimbine 1 microM and timolol 1 microM were examined. Subsequently, the vessels were preconstricted with NE to 60% of their maximal response and relaxation to acetylcholine 1 nM-0.1 mM was observed. Dietary n-3 PUFA supplements led to attenuation of the contractile responses of isolated resistance arteries (p < 0.01, repeated-measures analysis of variance, ANOVA-RM) versus control. The n-6 PUFA diet did not exert this effect although there was a downward trend. Diet did not affect EC50 values for NE. Neither n-3 nor n-6 PUFA diet influenced relaxation responses. The fatty acid composition of myocardial phospholipid fractions was significantly altered by both diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic adrenoceptors mediate sympathoadrenal activity in exercising rats.

The role of hypothalamic adrenoceptors in the exercise-induced alterations of plasma norepinephrine (NE), epinephrine (E), and corticosterone concentrations was investigated in rats. Exercise consisted of strenuous swimming against a counter-current for 15 min. Before, during, and after swimming, blood samples were withdrawn through a permanent heart catheter for determination of E, NE, and corticosterone. In control rats E, NE, and corticosterone levels were all increased during exercise. Infusion of the alpha-adrenoceptor antagonist phentolamine through permanent bilateral cannulas into the ventromedial hypothalamus (VMH) immediately before exercise reduced the exercise-induced increase in plasma E without affecting NE. Infusion of the beta-adrenoceptor antagonist timolol into the VMH enhanced plasma E and attenuated plasma NE increases. Infusion of phentolamine into the lateral hypothalamic area (LHA) led to enhanced NE and unchanged E concentrations, whereas infusion of timolol into the LHA caused a potentiation of the increase in plasma E without an effect on NE. Plasma corticosterone concentrations were not affected. The results suggest that 1) alpha- and beta-adrenoceptors in the hypothalamus influence peripheral catecholamine release, and 2) E and NE responses to exercise can be dissociated by interference of the central nervous system.

Immunopharmacological studies on Picrorhiza kurroa Royle-ex-Benth. Part IV: Cellular mechanisms of anti-inflammatory action.

In this work abroagation of anti-inflammatory effect of Picrorhiza kurroa extract (PK) by beta-adrenergic blockade was confirmed, which suggests alteration in cell-surface biology by PK treatment. Blockade of protein synthesis by cycloheximide pretreatment reduced PK effect, suggesting protein mediation. Metabolic inhibitor dinitrophenol inhibited inflammatory cedema equally in control and PK treated animals, and masking of PK effect was concluded. Discriminations of anti-inflammatory mechanism(s) of PK and the latter two cytotoxic agents was inferred from these observations and from existing knowledge. Selective PK influence on membrane linked activation events in inflammatory effector cells could be the basis of anti-inflammatory and perhaps other biological activities reported with the herb.

Central and peripheral adrenoceptors affect glucose, free fatty acids, and insulin in exercising rats.

The effects of intravenously and intrahypothalamically administered alpha- and beta-adrenoceptor antagonists on exercise-induced alterations in blood glucose, plasma free fatty acids (FFA), and insulin were investigated in rats. Exercise consisted of strenuous swimming against a counter current for 15 min. Before, during, and after swimming, blood samples were withdrawn through a permanent heart catheter. Intravenous administration of the alpha-blocker phentolamine led to a reduction in glucose and a substantial increase in insulin levels. Infusion of phentolamine through permanent bilateral cannulas into either the ventromedial or lateral area of the hypothalamus (VMH and LHA, respectively) completely prevented the increase in glucose while the decline in insulin was unaffected. Infusion of phentolamine into the VMH caused much higher plasma FFA levels than in controls. The beta-blocker timolol given intravenously caused a delayed increase in glucose and prevented the increase in FFA. Infusion of timolol into either VMH or LHA caused a delay in the increase in both glucose and FFA. The results suggest that 1) both peripheral and hypothalamic adrenoceptors are involved in energy metabolism during exercise and 2) FFA, glucose, and insulin concentrations in blood are independently regulated by VMH and LHA.

Acetylcholine and norepinephrine stimulate the release of corticotropin-releasing factor-41 from the rat hypothalamus in vitro.

The effects of the two putative neurotransmitters acetylcholine and norepinephrine on immunoreactive CRF-41 release from incubated rat hypothalami were studied. Acetylcholine at concentrations of 10(-11) to 10(-7) M stimulated CRF-41 release. This effect was blocked in a dose-dependent manner by the muscarinic antagonist atropine (10(-9) to (-7) M). The nicotinic antagonist hexamethonium was ineffective at a dose of 10(-7) M, but produced slight inhibition of this response at 10(-5) M. Norepinephrine at concentrations of 10(-10) to 10(-6) M also produced a dose-dependent stimulation of CRF-41 release. The beta-adrenoceptor antagonists propranolol (10(-5) M) and timolol (10(-6) M) blocked norepinephrine-induced CRF-41 release. The alpha 1-adrenoceptor antagonists thymoxamine (10(-5) M), prazosin (10(-5) M), and corynanthine (10(-4) M), and the alpha 2-antagonist idazoxan (10(-5) M), were ineffective. Potassium depolarization (56 mM) caused stimulation of CRF-41 release which was dependent on the presence of calcium in the incubation medium. Authenticity of immunoreactive CRF-41 released was demonstrated by chromatographic criteria using gel filtration and reversed phase HPLC. These results provide evidence for a stimulatory role of acetylcholine and norepinephrine on CRF-41 release, and consequently on hypothalamo-pituitary-adrenal axis in the rat, through actions at a hypothalamic level. The stimulatory effect of acetylcholine is mediated principally through muscarinic receptors and that of norepinephrine through beta-adrenoceptors.

Regional ocular blood flow after chronic topical glaucoma drug treatment.

The effects of a chronic three times a day treatment over a 5 to 6 week period of rabbit eyes with 1 of 5 topically applied glaucoma drugs on ocular blood flow was determined using a radioactive microsphere technique. The drugs employed were timolol (0.5%), pilocarpine (4%), epinephrine (2%), norepinephrine (2%), and ecothiophate iodide 0.125%). The results showed that epinephrine statistically decreased blood flow to the iris and ciliary processes while not to the posterior uveal tissues or optic nerve head. Pilocarpine also showed this same trend, while the differences were not statistically significant. Other drugs were without effect on regional ocular blood flow.

Effects of antiglaucoma drugs on the blood flow in rabbit eyes.

Although it is essential that intraocular pressure (IOP) be reduced in glaucoma treatment, it is also vitally important to provide sufficient blood flow to eye tissues so that healthy visual field is maintained. It is possible for an agent to reduce IOP and blood supply to the eye. In that case, glaucoma appears to be under control since IOP has been reduced to within normal range, yet the disease is actually progressing, causing damage to the retina, optic nerve, and other tissues. The 85Sr-microsphere technique was used to study the effects of several antiglaucoma drugs on blood supply to various eye tissues. It was found that pilocarpine, L-timolol, D-timolol and haloperidol are good drugs to use in treating glaucoma because they do not reduce ocular blood flow. D-timolol is particularly good because it does not cause side effects through beta-adrenergic blockade or cholinergic stimulation. On the other hand, trifluperidol and moperone reduce IOP effectively, but also decrease blood supply.