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.

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.

The mitochondrial Ca2+-independent phospholipase A(2) is activated during energy-dependent Ca2+ 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 Ca2+-independent phospholipase is a factor in the mechanisms by which depolarization and Ca2+ accumulation promote opening of the permeability transition pore. Sustained activity of the Ca2+-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 Ca2+-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.

RATIONALE: To explore loss of phosphatidylserine (PS) membrane asymmetry as part of the lytic mechanism of Ketotifen, Azelastine and Epinastine membrane interactions compared to the non-lytic Olopatadine.METHODS: Changes in PS plasma membrane asymmetry were determined by FACS analysis using fluorescein isothiocyanate (FITC) tagged annexin V. Enzymatic relocation of PS from the outer to the inner membrane leaflet was prevented by N-ethylmaleimide (NEM). Membrane leakage was quantified by hemoglobin release or by the appearance of a FTIC-annexin V labeled cell populationRESULTS: Exposure of NEM-treated erythrocytes to Olopatadine (0.1-10 mM) did not affect cell particle size, membrane permeability, or PS membrane localization. Ca2+- ion and ionophore (A23187) addition to NEM-treated erythrocytes (positive control) yielded a small population of permeabilized cells with changed particle size, in addition to an intact cell population that exhibited FITC-annexin V binding due to PS translocation to the outer membrane. Exposure of NEM-treated cells to Ketotifen, and Azelastine revealed erythrocyte permeabilization without translocation of PS to the outer membrane leaflet prior to cell lysis. Epinastine induced a significant transfer of PS from the inner to the outer membrane leaflet at concentrations below those causing cell permeabilization.CONCLUSIONS: Ketotifen and Azelastine cause erythrocyte membrane permeabilization without a prior disturbance of membrane PS asymmetry. Epinastine induces a significant disturbance in PS membrane asymmetry at sub-lytic concentrations. Oloptadine is totally devoid of membrane perturbing effects. Disturbance of membrane phospholipid asymmetry may be relevant to topical ocular use since PS exposure on the outer membrane leaflet has been reported to target cells for apoptosis. RATIONALE: To explore loss of phosphatidylserine (PS) membrane asymmetry as part of the lytic mechanism of Ketotifen, Azelastine and Epinastine membrane interactions compared to the non-lytic Olopatadine. METHODS: Changes in PS plasma membrane asymmetry were determined by FACS analysis using fluorescein isothiocyanate (FITC) tagged annexin V. Enzymatic relocation of PS from the outer to the inner membrane leaflet was prevented by N-ethylmaleimide (NEM). Membrane leakage was quantified by hemoglobin release or by the appearance of a FTIC-annexin V labeled cell population RESULTS: Exposure of NEM-treated erythrocytes to Olopatadine (0.1-10 mM) did not affect cell particle size, membrane permeability, or PS membrane localization. Ca2+- ion and ionophore (A23187) addition to NEM-treated erythrocytes (positive control) yielded a small population of permeabilized cells with changed particle size, in addition to an intact cell population that exhibited FITC-annexin V binding due to PS translocation to the outer membrane. Exposure of NEM-treated cells to Ketotifen, and Azelastine revealed erythrocyte permeabilization without translocation of PS to the outer membrane leaflet prior to cell lysis. Epinastine induced a significant transfer of PS from the inner to the outer membrane leaflet at concentrations below those causing cell permeabilization. CONCLUSIONS: Ketotifen and Azelastine cause erythrocyte membrane permeabilization without a prior disturbance of membrane PS asymmetry. Epinastine induces a significant disturbance in PS membrane asymmetry at sub-lytic concentrations. Oloptadine is totally devoid of membrane perturbing effects. Disturbance of membrane phospholipid asymmetry may be relevant to topical ocular use since PS exposure on the outer membrane leaflet has been reported to target cells for apoptosis.

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.