Efficacy and pharmacokinetics of intravitreal non-steroidal anti-inflammatory drugs for intraocular inflammation.

OBJECTIVE: To determine the efficacy and pharmacokinetics of intraocularly delivered non-steroidal anti-inflammatory drugs in an animal model of ocular inflammation. METHODS: Lipopolysaccharide was injected into the vitreous of rabbit eyes to induce inflammation. Treated eyes were injected with 3 mg of ketorolac or 0.3 mg of diclofenac. Twenty-four hours later, total leucocyte concentrations and prostaglandin E2 concentrations were determined. For intraocular pharmacokinetics, 0.1 ml of ketorolac (3 mg) and 0.1 ml of diclofenac (0.3 mg) were injected into rabbit eyes. Reverse-phase high-performance liquid chromatography was used to analyse drug levels within the retina/choroid at 0.25 (15 min), 1, 2, 4, 24, and 48 h after injection. RESULTS: Eyes treated with ketorolac and diclofenac demonstrated reduced aqueous leucocyte concentrations of 62% and 64% respectively, compared with untreated controls (p<0.05). Ketorolac and diclofenac reduced aqueous prostaglandin E2 levels by 85% (p<0.005) and 59% (p<0.005), respectively. Ketorolac and diclofenac achieved a peak vitreous concentration of 234 and 73 microg/ml, respectively. After 48 h, ketorolac was barely detectable (0.06 microg/ml) in the vitreous, and diclofenac was undetectable. The peak concentration of each drug in the retina/choroid was 201 microg/g for ketorolac and 4.1 microg/g for diclofenac. Both drugs were undetectable in the retina/choroid after 48 h. CONCLUSIONS: Both ketorolac and diclofenac have potent anti-inflammatory effects after intraocular injection. Pharmacokinetic analysis demonstrated good penetration into the retina/choroid but rapid clearance by 48 h.

Neural roles for heme oxygenase: contrasts to nitric oxide synthase.

The heme oxygenase (HO) and nitric oxide (NO) synthase (NOS) systems display notable similarities as well as differences. HO and NOS are both oxidative enzymes using NADPH as an electron donor. The constitutive forms of the enzyme are differentially activated, with calcium entry stimulating NOS by binding to calmodulin, whereas calcium entry activates protein kinase C to phosphorylate and activate HO2. Although both NO and carbon monoxide (CO) stimulate soluble guanylyl cyclase to form cGMP, NO also S-nitrosylates selected protein targets. Both involve constitutive and inducible biosynthetic enzymes. However, functions of the inducible forms are virtual opposites. Macrophage-inducible NOS generates NO to kill other cells, whereas HO1 generates bilirubin to exert antioxidant cytoprotective effects and also provides cytoprotection by facilitating iron extrusion from cells. The neuronal form of HO, HO2, is also cytoprotective. Normally, neural NO in the brain seems to exert some sort of behavioral inhibition. However, excess release of NO in response to glutamate's N-methyl-d-aspartate receptor activation leads to stroke damage. On the other hand, massive neuronal firing during a stroke presumably activates HO2, leading to neuroprotective actions of bilirubin. Loss of this neuroprotection after HO inhibition by mutant forms of amyloid precursor protein may mediate neurotoxicity in Familial Alzheimer's Disease. NO and CO both appear to be neurotransmitters in the brain and peripheral autonomic nervous system. They also are physiologic endothelial-derived relaxing factors for blood vessels. In the gastrointestinal pathway, NO and CO appear to function as coneurotransmitters, both stimulating soluble guanylyl cyclase to cause smooth muscle relaxation.

Atypical neural messengers.

Notions of what constitutes a neurotransmitter have changed markedly with the advent in the past decade of synaptic molecules, which satisfy key neurotransmitter criteria but differ radically from classical transmitters. Thus, NO and carbon monoxide are neither stored in synaptic vesicles nor released by exocytosis. These gases do not act via traditional receptors on postsynaptic membranes. In addition, zinc, stored together with glutamate in synaptic vesicles, appears to act as an 'antagonist' co-transmitter at the NMDA receptor, and although localized exclusively to glia, D-serine fulfills most neurotransmitter criteria as an endogenous ligand for the 'glycine' site of NMDA receptors.

A mammalian iron ATPase induced by iron.

While molecular mechanisms for iron entry and storage within cells have been elucidated, no system to mediate iron efflux has been heretofore identified. We now describe an ATP requiring iron transporter in mammalian cells. (55)Fe is transported into microsomal vesicles in a Mg-ATP-dependent fashion. The transporter is specific for ferrous iron, is temperature- and time-dependent, and detected only with hydrolyzable nucleotides. It differs from all known ATPases and appears to be a P-type ATPase. The Fe-ATPase is localized together with heme oxygenase-1 to microsomal membranes with both proteins greatly enriched in the spleen. Iron treatment markedly induces ATP-dependent iron transport in RAW 264.7 macrophage cells with an initial phase that is resistant to cycloheximide and actinomycin D and a later phase that is inhibited by these agents. Iron release, elicited in intact rats by glycerol-induced rhabdomyolysis, induces ATP-dependent iron transport in the kidney. Mice with genomic deletion of heme oxygenase-1 have selective tissue iron accumulation and display augmented ATP-dependent iron transport in those tissues that accumulate iron.

Haem oxygenase-1 prevents cell death by regulating cellular iron.

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.