Differential effects of non-steroidal anti-inflammatory drugs on mitochondrial dysfunction during oxidative stress.

We investigated the effects of several non-steroidal anti-inflammatory drugs on swelling related properties of mitochondria, with an emphasis on compounds that are marketed and utilized topically in the eye (nepafenac, ketorolac, diclofenac, bromfenac), and compared these to the effects of amfenac (a metabolite of nepafenac) and to celecoxib (active principle of Celebrex). With the exception of the last compound, none of the drugs promote swelling of normal mitochondria that are well energized by succinate oxidation. However, swelling is seen when the mitochondria are under an oxidative stress due to the presence of t-butylhydroperoxide. When used at 200 microM the order of potency is celecoxib > bromfenac > diclofenac > ketorolac > amfenac > nepafenac approximately equal to 0. Again with the exception of celecoxib, this swelling is not seen when mitochondria are depleted of endogenous Ca(2+) and is accelerated when exogenous Ca(2+) is provided. Sr(2+) does not substitute for exogenous Ca(2+) and prevents swelling in the presence of endogenous Ca(2+) only. The same is true for ruthenium red (inhibitor of the Ca(2+) uniporter), for cyclosporin A (inhibitor of the mitochondrial permeability transition), and for a 3.4 kDa polyethylene glycol (polymer that cancels the force which drives swelling following the permeability transition). It is concluded that several non-steroidal anti-inflammatory drugs promote the mitochondrial permeability transition under conditions of oxidative stress and in a Ca(2+) dependent fashion, whereas celecoxib functions by another mechanism. Potency of those compounds that promote the transition varies widely with bromfenac being the most potent and nepafenac having almost no effect. The mitochondrial dysfunction which is caused by the transition may underlie side effects that are produced by some of these compounds.

Comparative effects of the nonsteroidal anti-inflammatory drug nepafenac on corneal sensory nerve fibers responding to chemical irritation.

PURPOSE: To compare the corneal analgesic efficacy of the nonsteroidal anti-inflammatory drugs (NSAIDs) nepafenac, diclofenac, and ketorolac, and to evaluate the possibility that their inhibitory effects on corneal polymodal nociceptor fiber activity are partly mediated by a decrease in sodium currents. METHODS: Corneal sensory afferent units were recorded in the anesthetized cat. The response of thin myelinated polymodal nociceptor fibers to mechanical and acidic stimulation (98.5% CO(2)) was recorded before and at various times after topical application of the vehicle or of nepafenac 0.1% (Nevanac; Alcon Laboratories, Ltd., Fort Worth, TX), diclofenac 0.1% (Voltaren; Novartis, Basel, Switzerland), and ketorolac 0.4% (Acular LS; Allergan, Irvine, CA). Voltage-clamp recordings were performed in cultured trigeminal ganglion neurons. RESULTS: Nepafenac, diclofenac, and ketorolac reduced the mean frequency of the impulse response evoked by repeated CO(2) stimuli in polymodal nociceptor fibers. The progressive increase in ongoing activity, observed in vehicle-treated eyes after repeated acidic stimulation was also prevented. Nepafenac exhibited a more rapid and a slightly more pronounced effect on spontaneous and CO(2)-evoked activity than did diclofenac and ketorolac and did not affect the responsiveness of corneal mechanonociceptor or cold receptor fibers. In cultured mice trigeminal ganglion neurons, diclofenac significantly suppressed sodium currents, whereas nepafenac or its metabolite, amfenac, exhibited only minimal inhibitory effects. CONCLUSIONS: The inhibition of polymodal nociceptor activity by nepafenac, a weak inhibitor of cyclooxygenase, is most likely due to its greater lipophilicity compared with diclofenac and ketorolac, leading to a rapid saturation of the corneal epithelium where nociceptor terminals are located. In contrast to diclofenac, nepafenac does not exhibit local anesthetic effects.

Mitochondrial iPLA2 activity modulates the release of cytochrome c from mitochondria and influences the permeability transition.

The mitochondrial Ca(2+)-independent phospholipase A(2) is activated during energy-dependent Ca(2+) accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca(2+)-independent phospholipase is a factor in the mechanisms by which depolarization and Ca(2+) accumulation promote opening of the permeability transition pore. Sustained activity of the Ca(2+)-independent phospholipase A(2) promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca(2+)-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.

AL-2512, a novel corticosteroid: preclinical assessment of anti-inflammatory and ocular hypertensive effects.

The anti-inflammatory efficacy and ocular hypertensive effect of AL-2512 were characterized in rodent and feline models of ocular inflammation. Neutrophil influx into ocular tissue following topical ocular administration of test drugs was evaluated in models of endotoxin-induced uveitis. In rats, the anti-inflammatory efficacy of AL-2512 was compared with that of 0.1% dexamethasone. Test drug or vehicle was administered topically before subplantar injection of endotoxin. Neutrophil influx was assessed at 24 hours. Feline eyes, injected intravitreally with endotoxin, were treated topically with 0.1% AL-2512, 1.0% prednisolone acetate or vehicle at various timepoints before and after endotoxin injection. At 12 hours, protein concentration and leukocyte count in aqueous humor were determined. In the feline intraocular pressure (IOP) model, after baseline IOP values were established, AL-2512, dexamethasone, or vehicle was administered topically to both eyes of cats. IOP was measured daily before and during treatment. Topical ocular administration of AL-2512 inhibited endotoxin-induced leukocyte influx in rodent and feline models of uveitis. In rats, AL-2512 significantly inhibited neutrophil influx by 89%, compared with 93% by dexamethasone. In feline eyes, AL-2512 significantly (p < 0.05) inhibited leukocyte infiltration of aqueous humor by 59%, compared to 37% inhibition by prednisolone acetate. Intraocular pressure in cats treated for 32 days with AL-2512 or dexamethasone increased 6% and 18%, respectively. The ocular anti-inflammatory effect of AL-2512 was equivalent to dexamethasone and superior to prednisolone acetate in rat and feline models of ocular inflammation, respectively. This steroid provides anti-inflammatory efficacy equivalent to dexamethasone with a reduced risk of inducing ocular hypertension.

The hydrolysis of the prostaglandin analog prodrug bimatoprost to 17-phenyl-trinor PGF2alpha by human and rabbit ocular tissue.

Bimatoprost (Lumigan), the ethyl amide derivative of the potent prostaglandin FP agonist 17-phenyl-trinor PGF(2alpha), has been reported to be a member of a pharmacologically unique class of ocular hypotensive agents. To confirm that bimatoprost, which is intrinsically active as an FP prostaglandin agonist, is also a prostaglandin analog prodrug, the hydrolysis of bimatoprost by ocular tissues was studied by incubating solutions containing bimatoprost with either human or rabbit ocular tissue. The ethyl amide group of bimatoprost was hydrolyzed by rabbit and human cornea, iris/ciliary body and Thasclera to produce the expected carboxylic acid product, 17-phenyl-trinor PGF(2alpha). The rate of hydrolysis by human and rabbit cornea and iris/ciliary body is similar, whereas the rate of hydrolysis by the sclera is slower in humans than in rabbits. These studies show that human and rabbit ocular tissue (cornea, iris/ciliary body and sclera) can convert bimatoprost to the potent prostaglandin FP agonist 17-phenyl-trinor PGF(2alpha). Separate in vitro studies clearly show that both bimatoprost and 17-phenyl-trinor PGF(2alpha) have affinity for and are agonists at the human FP receptor. Taken together, the data strongly suggests that the ocular hypotensive effect of bimatoprost can be attributed to its activity as a prostaglandin receptor agonist either directly or through its role as a prostaglandin agonist prodrug.

Characterization of the in vitro anti-inflammatory activity of AL-5898 and related benzopyranyl esters and amides.

Selected ester- (AL-5898 and AL-8417) and amide-linked benzopyran analogues (AL-7538 and AL-12615) were evaluated in vitro for their ability to inhibit key enzymes/processes of the inflammatory response. AL-7538 and AL-12615 exhibited weak intrinsic cyclooxygenase inhibitory activity (IC50 = 13 microM, 37 microM). In contrast, 5-HETE and LTB4 synthesis in A(23187)-stimulated neutrophils was effectively inhibited by both ester and amide analogs (IC50 = 2-3 microM). While there was some indication for differing sensitivities among benzopyran esters and amides in the suppression of cytokine synthesis in stimulated U-937 cells, there appeared to be no great discrimination when assessing their effect on U-937 cell adhesion to IL-1beta activated HMVEC-L cells. Inhibition of cell adhesion was concentration-dependent, with IC50 values ranging between 18 microM and 30 microM for AL-5898. Concentration-dependent inhibition of inflammatory cytokine production (i.e., IL-1beta, TNF-alpha, GM-CSF and IL-6) was also apparent in LPS-stimulated, cultured PBMC as well as in PMA/A(23187) activated U-937 cells monitoring the synthesis of IL-1beta, IL-8, TNF-alpha, and MCP-1. Notably, the hydrolysis products of the benzopyranyl ester, AL-5692 and (S)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid, were devoid of pharmacological activity when assessed for inhibition of monocyte adhesion or IL-1beta synthesis. Collectively, our data demonstrate the unique in vitro polypharmacology of a novel series of benzopyran analogs that suppress pivotal enzymes and processes in the inflammatory response.

Interactions of olopatadine and selected antihistamines with model and natural membranes.

OBJECTIVE: Olopatadine, an effective topical ocular human conjunctival mast cell stabilizer/antihistaminic antiallergic drug, was evaluated and compared to selected classical antihistamines for their interaction with model and natural membranes to ascertain potential functional consequences of such interactions. METHODS: The model membranes examined consisted of the argon-buffer interface and monomolecular films of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) at the argon-buffer interface. Interactions with the model membranes were detected as changes in surface tension, i.e., surface pressure. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage, hemoglobin release, lactate dehydrogenase release, and histamine release from appropriate cell types. RESULTS: Measurements at the argon-buffer interface revealed intrinsic surface activity for all agents that ranged from highly surface-active to weakly surface-active in the order of: desloratadine > clemastine > azelastine congruent with ketotifen > diphenhydramine> pyrilamine > emedastine > epinastine > or = olopatadine. This order of amphipathic behavior was confirmed for most of the compounds by estimates of their dissociation constants (K(d,L)) determined from interactions with SOPC monolayers adjusted to a surface pressure approximating that of natural membranes. Epinastine was the only antihistamine that showed a disproportionately greater increase in surface activity toward SOPC in monolayer when compared to other antihistamines. Dissociation constants could not be established for olopatadine because of its low affinity for both the argon-buffer interface and the SOPC monolayer. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage (erythrocyte ghosts), hemoglobin release (erythrocytes), lactate dehydrogenase release (conjunctival mast cells, corneal epithelial cells), and histamine release (conjunctival mast cells). Aside from olopatadine and emedastine, all antihistamines promoted a concentration-dependent leakage of hemoglobin from intact erythrocytes. The concentration of drug required to cause half-maximal hemoglobin release (H(50)) from erythrocytes correlated linearly (r = 0.98) with the SOPC dissociation constants (K( d,L)) estimated for the different antihistaminic agents interacting with SOPC monolayers. A similarly high correlation (r = 0.85) emerged from a plot with a slope approaching unity that related drug concentrations required for half-maximal hemoglobin leakage from erythrocytes to threshold doses of drug that caused histamine release from human conjunctival mast cells. Olopatadine was the only agent that did not promote membrane perturbation as monitored by either hemoglobin release from intact erythrocytes, LDH release from human conjunctival mast cells, or 6-carboxyfluorescein release from erythrocyte ghosts. Assessment of the lytic potential of marketed concentrations of ketotifen (0.025%), azelastine (0.05%), and epinastine (0.05%) revealed significant membrane perturbation of human conjunctival mast cells and, importantly, human corneal epithelial cells as indexed by LDH release. This was in contrast to marketed concentrations of olopatadine (0.1%) which maintained normal mast cell and corneal epithelial cell membrane function. CONCLUSIONS: Combined, these results support the notion that the disruption of natural cell membranes by surface-active antihistamines occurs not through a receptor-mediated process, but is the consequence of a direct interaction of these agents with the cell membrane. This is corroborated by surface pressure-concentration isotherms for adsorption of five different antihistaminic agents to SOPC monolayers where 50% lysis occurred at a surface pressure of 42.9 +/- 1.1 mN/m. Olopatadine appears to be unique among the agents tested by demonstrating low intrinsic surface activity, thus limiting its interaction with natural membranes. At concentrations of about half-maximal compound solubility (, 5.0 mM or a 0.19% drug solution), olopatadine generated SOPC monolayer surface pressures (i.e., 39.82 +/- 0.10 mN/m) that were below those that promoted membrane perturbation and onset of hemoglobin leakage. Olopatadine's restricted interaction with membrane phospholipids limits the degree of membrane perturbation and release of intracellular constituents, including histamine, LDH, and hemoglobin, which is believed to contribute to olopatadine's topical ocular comfort and patient acceptance.