Receptor tyrosine kinase inhibitors AG013764 and AG013711 reduce choroidal neovascularization in rat eye.

Age-related macular degeneration (AMD) is the major cause of blindness for people over 60. In the "wet" form of AMD compounds targeting growth factor signaling pathways such as VEGF have been a major focus for therapeutic interventions. In a previously developed rat model of CNV, we utilized two receptor tyrosine kinase inhibitors (RTKi) to block VEGFR-1, VEGFR-2 and PDGFR signaling following the establishment of CNV. AAV-VEGF(165) was injected into the subretinal space of rats at postnatal days 15-17. Six weeks later, a suspension of RTK inhibitors, AG013764 or AG013711, was injected intraperitoneally (IP, twice daily) or intravitreally (every five days) over a two week period. FITC-dextran whole-mounts of RPE-choroid-sclera were prepared after the animals were sacrificed. CNV area was quantified using Neurolucida to measure the hyperfluorescence on FITC-dextran whole-mounts. Histology and immunohistochemistry were performed as described previously. VEGF expression in control and treated eyes was confirmed by immunohistochemistry and histological sections indicated recovery of retinal morphology and CNV reduction in treated eyes. In the animals IP injected with AG013764 or AG013711 the mean CNV level was reduced by 25 to 33% compared to control, but this effect did not achieve statistical significance. Intravitreal injections of AG013764 or AG013711 reduced the level of CNV by approximately 60% compared to control (p<0.005 or p<0.05, respectively). These data show that two RTK inhibitors, AG013764 or AG013711, delivered intravitreally, significantly reduce blood vessel proliferation in this AAV-VEGF(165) model of CNV.

Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species.

Hepatotoxic doses of acetaminophen to mice produce not only acetaminophen-protein adducts in the centrilobular cells of the liver, but nitrotyrosine-protein adducts in the same cells, the site of the necrosis. Nitration of tyrosine occurs with peroxynitrite, a species formed by reaction of nitric oxide (NO.) with superoxide (O2. -). Because NO. and O2.- may be produced by activated Kupffer cells and/or infiltrated macrophages, we pretreated mice with the macrophage inactivators/depeleters gadolinium chloride (7 mg/kg, intravenously [iv]) or dextran sulfate (10 mg/kg, iv) 24 hours before administration of acetaminophen (300 mg/kg). Mice treated with acetaminophen plus gadolinium chloride, or acetaminophen plus dextran sulfate, had significantly less evidence of hepatotoxicity as evidenced by lower serum alanine transaminase (ALT) levels (28 +/- 1 IU/L and 770 +/- 240 IU/L, respectively) at 8 hours compared with acetaminophen (6,380 +/- 408 IU/L). Analysis of hepatic homogenates for acetaminophen-protein adducts at 2 hours, a time of maximal covalent binding and before hepatocyte lysis, indicated that these pretreatments did not decrease covalent binding. Western blot analysis for the macrophage marker protein F4/80 in homogenates revealed not only the expected decrease by the macrophage inactivators/depleters, but also an apparent increase in acetaminophen-only-treated mice. At 8 hours nitrotyrosine-protein adducts were present in the acetaminophen-only-treated mice, but not in the acetaminophen plus gadolinium chloride-treated mice, or acetaminophen plus dextran sulfate-treated mice. High levels of heme-protein adducts, a measure of oxidative stress, were detected in livers of the 8 hour acetaminophen-only-treated mice. These data suggest that acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding, and subsequent activation of macrophages to form O2.-, NO., and peroxynitrite. Nitration of tyrosine correlates with toxicity.

Liposome uptake by human retinal pigment epithelial cells in culture.

PURPOSE: To examine the internalization of liposomes containing various phospholipids by human retinal pigment epithelial (RPE) cells in culture. The internalization was compared to that exhibited by macrophages and fibroblast cells. METHODS: The uptake into cells was monitored by using a non-degradable, radioactive cholesterol-hexadecyl ether derivative and a pH-sensitive, water-soluble fluorescent dye. Variables tested to determine their effect on uptake included time, phospholipid concentration, the presence or absence of serum in medium and the presence or absence of cholesterol in the liposome bilayer. The most avidly ingested liposome was tested to determine its ability to deliver a liposome-dependent drug. RESULTS: Liposome uptake was time and concentration dependent. Uptake for RPE was maximal in serum-free media, while the opposite was true for the other cells tested. Cell survival following exposure to fluoroorotic acid-containing liposomes correlated with the radioactive cholesterol-hexadecyl ether and fluorescent dye uptake data. CONCLUSIONS: No single liposome carrier or set of conditions tested appeared to be optimum for the delivery of a cytotoxic agent to these three cell types. This bears consideration when designing strategies for the prevention and treatment of intraocular proliferative diseases such as proliferative vitreoretinopathy (PVR).

Therapeutic effect of liposomal superoxide dismutase in an animal model of retinopathy of prematurity.

A newborn rat model of retinopathy of prematurity was used to test the hypothesis that a lack of superoxide dismutase contributes to the retinal vaso-attenuation seen during exposure of the animals to hyperoxic conditions. To determine the endogenous superoxide dismutase activity of the retina under hyperoxic conditions, litters of albino rats were placed in either constant 80% ambient oxygen (constant hyperoxia), or placed in 21% oxygen (room air) immediately after birth. Every other day, for 14 days, several rat pups were sacrificed and their retinas removed for the determination of total superoxide dismutase (SOD) activity and manganese-associated SOD activity. An attempt was made to increase retinal SOD activity by intraperitoneal administration of exogenous SOD encapsulated in polyethylene glycol-modified liposomes. Additional litters were exposed to the same oxygen treatments and supplemented twice daily with either liposome-encapsulated superoxide dismutase in saline or liposomes containing saline without SOD. Animals were sacrificed at various time points for the determination of total superoxide dismutase activity and computer-assisted analysis of vessel density and avascular area. Animals raised in an atmosphere of constant 80% oxygen had significantly reduced levels of retinal superoxide dismutase activity through 6 days of life when compared to their room air-raised littermates. At 6 days of age, daily supplementation with liposome-encapsulated SOD had significantly increased retinal superoxide dismutase activity and reduced oxygen-induced vaso-attenuation as evidenced by increased vessel density and decreased avascular area, when compared to littermates exposed to constant hyperoxia that received control liposomes. Superoxide dismutase had no adverse effects on any of the animals regardless of treatment. Tracing experiments demonstrated that liposomes entered the retina and were found in cells morphologically resembling microglia. Delivery of SOD to the retina via long-circulating liposomes proved beneficial, suggesting that restoration and/or supplementation of endogenous antioxidants in oxygen-damaged retinal tissue is a potentially valuable therapeutic strategy.

Vancomycin levels in the vitreous cavity after intravenous administration.

PURPOSE: We studied the effects of inflammation, repeated antibiotic doses, and the surgical status of the eye on penetration of vancomycin hydrochloride into the rabbit vitreous cavity after intravenous administration. METHODS: We studied three anatomic states (phakic, aphakic, and aphakic, vitrectomy-treated eyes) subdividing each into inflamed and noninflamed groups. Intravenous vancomycin hydrochloride (15 mg/kg of body weight) was administered every 12 hours for 48 hours. Eyes were harvested for the assay of vitreous cavity antibiotic levels at various intervals from one to 49 hours. We determined concentrations and calculated mean values and S.E.M. RESULTS: Therapeutic levels were not established in the vitreous cavity at any time period in the two phakic groups. At 25 hours, the inflamed aphakic eyes had concentrations of 5.05 +/- 1.9 micrograms/ml and the control noninflamed aphakic eyes 4.5 +/- 1.23 micrograms/ml; slight increases were found by 49 hours. Concentrations tested in the aphakic, vitrectomy-treated eyes at two, 13, 25, and 49 hours demonstrated progressive increases both in the inflamed eyes (5.4 +/- 2.4 micrograms/ml, 9.64 +/- 4.25 micrograms/ml, 9.2 +/- 3.96 micrograms/ml, 10.34 +/- 4.49 micrograms/ml) and noninflamed eyes (3.52 +/- 2.1 micrograms/ml, 5.4 +/- 1.96 micrograms/ml, 6.8 +/- 2.53 micrograms/ml, 8.7 +/- 5.44 micrograms/ml). CONCLUSIONS: Vitreous vancomycin concentrations in aphakic and aphakic, vitrectomy-treated eyes after intravenous administration exceed the minimal inhibitory concentrations for the usual gram-positive pathogens that create endophthalmitis, suggesting a role for intravenous vancomycin in the treatment of bacterial endophthalmitis.

Shiva-1: in vitro and in vivo tests of the effects of a novel, synthetic, lytic peptide on ocular cells.

An investigation was undertaken to determine the toxicity of an intravitreal injection of a novel peptide drug, Shiva-1, in rabbits. The drug, a synthetic peptide modeled after lytic peptides secreted by certain insects, has antiproliferative and antibacterial properties. Initial in vitro experiments showed that the drug, at a concentration of 100 microM, was toxic to both Y-79 retinoblastoma cells and human retinal pigment epithelial cells. A wide range of doses (6-1200 micrograms) was injected into the rabbit vitreous in an attempt to determine the maximum tolerated dose. Retinal toxicity was evaluated clinically, by electroretinography, and by light microscopy. Some localized toxicity was evident at 200 micrograms; all doses of 240 micrograms and above were toxic. While the drug appears to exhibit a narrow range between effective and toxic doses, the results suggest that this and other peptides of similar design merit further investigation for the treatment of proliferative and infectious diseases of the eye.

Modulation of hepatic sinusoidal endothelial cell function by Kupffer cells: an example of intercellular communication in the liver.

We tested the hypothesis that Kupffer cells modulate sinusoidal endothelial cell function in the liver. Rats were treated with Kupffer cell-depleting agents (gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate) or with inhibitors of phospholipase A2 or leukotriene A4 synthase (dexamethasone and diethylcarbamazine, respectively). Hyaluronan uptake by the isolated, perfused liver was measured as an index of the functional state of the sinusoidal endothelial cell. Plasma hyaluronan concentration was also determined. Three hours after Escherichia coli lipopolysaccharide administration (100 micrograms/100 gm body wt, intravenously) plasma hyaluronan levels were significantly increased (280% to 320%), whereas hepatic hyaluronan uptake was markedly decreased (approximately 76%). Pretreatment with gadolinium chloride (0.5 mg/100 gm body wt, intravenously, 21 hr before saline solution or lipopolysaccharide administration), liposome-encapsulated dichloromethylene diphosphonate (40 mumol/100 gm body wt, intravenously, 44 hr before saline solution or lipopolysaccharide injection), dexamethasone (40 micrograms/100 gm body wt, intravenously, 1 hr before saline solution or lipopolysaccharide administration) or diethylcarbamazine (repeated doses, 10 mg/100 gm body wt, intravenously, 1 hr before saline solution or lipopolysaccharide injection) counteracted the lipopolysaccharide inhibitory effect on hepatic hyaluronan uptake. With the exception of gadolinium chloride, all other agents also prevented the lipopolysaccharide-induced increase in plasma hyaluronan concentration. Gadolinium chloride only attenuated the lipopolysaccharide effect on plasma hyaluronan level. Taken together with earlier results from our laboratory, these data indicate that: (a) Kupffer cell activation by lipopolysaccharide results in suppression of hyaluronan uptake by sinusoidal endothelial cells and (b) such modulation of endothelial cell function is likely mediated by products of the lipoxygenase pathway of arachidonate metabolism.

Evaluation of toxicity and efficacy of a combination of antineoplastic agents in the prevention of PVR.

The retinal toxicity of a combination of antineoplastic drugs in free and liposome-encapsulated form was determined in the rabbit eye. Bleomycin sulfate and 5-fluorouridine were evaluated by clinical observation, electroretinogram, and histological study. Forty-five eyes were injected with combinations of various doses of bleomycin and 5-FUR in free and encapsulated form; 10 eyes served as controls. The nontoxic free dose was found to be 3.5 micrograms bleomycin and 150 micrograms 5-FUR. Liposome encapsulation increased the nontoxic dose to 4.7 micrograms bleomycin and 200 micrograms 5-FUR. Four groups of rabbits in which proliferative vitreoretinopathy had been induced were used for the efficacy study; the control group received an injection of PBS; the second group was injected with a combination of 3.5 micrograms bleomycin and 150 micrograms 5-FUR in free form; the third group was injected with the identical doses in liposome-encapsulated form; and the fourth group received encapsulated bleomycin (4.7 micrograms) and 5-FUR (200 micrograms). The dose used in Group 4 was significantly more effective (P < 0.01) in preventing tractional retinal detachment and marginally more effective (P = 0.054) in preventing neovascularization.

Encapsulation of sodium fluorescein for dye release studies.

Recent investigations have detailed a selective dye release technique in which a pulse of laser light induces the release of a fluorescent dye from temperature-sensitive liposomes circulating in the retinal vasculature. This dye release technique has made possible a new method for measuring ocular blood flow in the retina and has spurred the development of repetitive, site-specific angiography. However, sodium fluorescein, the dye employed clinically for angiography of the retina, has not been employed in the aforementioned studies because of its rapid efflux from liposomes. This report outlines a method for stable encapsulation of sodium fluorescein in temperature-sensitive liposomes. Heat-induced leakage of the dye from liposomes in vitro was similar to that previously seen with other fluorescent dyes. Furthermore, after intravenous injection of encapsulated fluorescein in a nonhuman primate, dye released by a pulse of laser light allowed excellent visualization of the retinal architecture. These results indicate that sodium fluorescein, a dye that has proven to be the agent of choice for sensitive detection of leakage of vessels of the retina, can be released at a specific site in the retinal vasculature. Direct comparisons of the diagnostic capability of free and encapsulated sodium fluorescein are now possible.

The use of liposomes as drug carriers in ophthalmology.

Extensive investigations examining the use of liposomes as drug carriers have been carried out over the past 25 years. The concept of employing liposomes as drug carriers in ophthalmology is more recent. A major advantage of employing liposomes in the eye is their ability to evade initially the mononuclear phagocytes that make the intravenous use of liposomes so challenging. Recent work suggests that there are several applications in which use of liposome-entrapped drugs may prove superior to other methods for the diagnosis and treatment of ocular disease. One promising strategy that bears further investigation is ligand-mediated targeting of liposome-entrapped drugs. In order for these types of vesicles to become commercially available, they will have to be proven to be more effective than other targeted drug delivery modalities. In the final analysis, a more complete understanding of the biological changes that occur in various pathological conditions in the eye is needed in order to design the most effective drug carriers for use in ophthalmology.

Dextran magnetite as a liver contrast agent.

The superparamagnetic particle dextran magnetite was studied as a liver tumor contrast agent for magnetic resonance imaging (MRI). The effects of dextran magnetite on the longitudinal (T1) and transverse (T2) relaxation times in liver, spleen, and an implanted rat liver tumor were measured at 0.47 T (IBM/Bruker PC-20 relaxometer) over the dose range of 23 to 69 mumol Fe/kg. Dextran magnetite substantially reduced the T2 of the liver and spleen, but not of the tumor, thereby providing a basis for improved tumor imaging. The T1 of the tumor was not affected following injection of dextran magnetite in the dose range studied, while the spleen T1 was reduced substantially more than the T1 of the liver. Histological studies using the iron reaction for Prussian blue clearly showed dextran magnetite in the liver and spleen, but not in the tumor. While dextran magnetite was sequestered in macrophages in both liver and spleen, the distribution in the liver was more diffuse (70 microns average particle separation) than that in the spleen (25 microns separation). The lack of a T1 effect in the liver is consistent with the fact that a majority of the water in the tissue cannot diffuse to the relaxational centers on the time scale of the liver's intrinsic T1 (280 ms). In the spleen, however, the dextran magnetite is more densely packed in the red pulp allowing a significant fraction of the water to be relaxed by a T1 mechanism. Spin-echo images of the implanted tumor (mammary adenocarcinoma. R3230AC) in the livers of Fischer 344 rats were obtained at 0.50 T (Siemens Magnetom). The tumor-to-liver contrast was improved for both T1 and T2-weighted spin-echo images after intravenous injection of the dextran magnetite contrast agent. The contrast determined from these images agreed with that predicted by the measured T1 and the T2 (Hahn spin-echo) values. In addition, gradient-echo T2-weighted images with good contrast were obtained in a much shorter imaging time than was needed for T2-weighted spin-echo images. These results demonstrate that the MRI contrast enhancement observed with dextran magnetite is based on its selective uptake and distribution in the macrophages in the liver and spleen and that this agent has substantial potential as a superparamagnetic MR contrast agent.

Ciprofloxacin and dexamethasone inhibit the proliferation of human retinal pigment epithelial cells in culture.

We investigated the inhibition of proliferation of human retinal pigment epithelial cells in vitro by the 4-quinolone, ciprofloxacin, and the steroid, dexamethasone. The concentration of ciprofloxacin that inhibited growth by 50% (IC50) was found to be 14.1 micrograms/mL. Growth was 100% inhibited at 83 micrograms/mL. At 166 micrograms/mL, all the cells became completely detached and appeared dead at the end of seven days. The IC50 for dexamethasone in RPE cells was found to be 141 micrograms/mL. A dexamethasone concentration of 1.3 mg/mL inhibited proliferation 100% after five days. When the two drugs were combined, the inhibitory effect was found to be additive; i.e., the IC50 dose of the two drugs in combination inhibited RPE cell proliferation by 75%. A combination of the two drugs was also tested for retinal toxicity in rabbit eyes. An examination of histological sections and electroretinograms showed that a dose of 100 micrograms of ciprofloxacin, alone or in combination with 200 micrograms of dexamethasone in saline, was not toxic to the rabbit retina. These studies indicate that a combination of ciprofloxacin and dexamethasone has the potential for reducing the risk of PVR formation and aiding in the prevention of endophthalmitis.

Retinal toxicity and in vitro efficacy study of cilofungin (LY121019).

We assessed the retinal toxicity of a new antifungal agent, cilofungin, by injecting it into the vitreous body of albino rabbit eyes in dosages of 2.5 micrograms to 320 micrograms. The maximum intravitreal drug concentration (320 micrograms) produced no histological evidence of retinal toxicity. Electroretinography data showed some variations, but no toxicity at the highest dose (320 micrograms). When efficacy was evaluated in vitro against Candida albicans, the minimal inhibitory concentration was 2.50 micrograms/mL, using Sabouraud's broth or M3 medium. The minimal fungicidal concentration was 5 micrograms/mL with Sabouraud's broth, and 10 micrograms/mL with M3 medium. Cilofungin is a potentially safe antifungal for the treatment of candida endophthalmitis.

Liposome encapsulated MnCl2 as a liver specific contrast agent for magnetic resonance imaging.

We examined the usefulness of MnCl2 entrapped in liposomes as a liver specific contrast agent for magnetic resonance imaging. Toxicity experiments showed that the effective dose for imaging experiments was 7 to 11 times lower than the LD50 of free MnCl2. In rats with implanted liver tumors, liposome encapsulated MnCl2 caused a two- to three-fold increase in the relaxation rate of the liver while having little effect on the relaxation rate of tumor tissue. T1 weighted, magnetic resonance images obtained at 0.5 T of an R3230 adenocarcinoma implanted in the liver showed an increase in the signal intensity of both normal liver and tumor tissue after the injection of free MnCl2 (25 mumole/kg). However, after the injection of liposome encapsulated MnCl2 (40 mumole/kg) the liver exhibited a marked increase in signal intensity with little change in the signal intensity of the tumor tissue. These results suggest that liposome encapsulated MnCl2 has excellent potential as a liver specific contrast agent for the improved detection of liver metastases.

NMR and ESR study of liposome delivery of Mn2+ to murine liver.

The mechanism of tissue relaxation of liposome-delivered Mn2+ as a contrast agent for magnetic resonance imaging (MRI) was examined using magnetic resonance and electron spin resonance (ESR) techniques. It is known that liposomes of the size and composition used in this study are taken up by fixed liver macrophages (Kupffer cells). It was determined that Mn2+ must be released from the liposomes in order to affect the water proton relaxation rate in the liver. As long as the Mn2+ was confined to the Kupffer cells, no substantial changes in the relaxation of the majority of the liver water were observed. Unlike other contrast agents delivered to the Kupffer cells (for example, Gd-starch microspheres or magnetite), once the Mn2+ is delivered and released into the Kupffer cells, it can diffuse from the Kupffer cells and be rapidly taken up by the hepatocytes. This seems to be the mechanism for selective relaxation enhancement in the liver. A consequence of this behavior is that the time at which maximum contrast enhancement occurs for MRI can be varied by the choice of liposome phospholipid composition. ESR techniques were used to directly determine the state of Mn2+ and the integrity of liposomes in various stages of processing.

Measurement of retinal blood velocity and flow rate in primates using a liposome-dye system.

A recently developed liposome-dye method was used to make quantitative measurements of blood velocity and volumetric flow rates in the retina of the rhesus monkey. After systemic injection of a liposome-encapsulated fluorescent dye (calcein), the release of a bolus of the dye in the retina was triggered by an ophthalmic argon laser. Initially, calcein was entrapped at a high concentration and the fluorescence of the dye in the lipid vesicle was quenched. After laser exposure, the liposomes released a bolus of dye at the laser exposure site in a specific vessel of the retina. The released dye formed a well-defined, fluorescent wavefront in the exposed artery or vein. By measuring the distance the dye traveled in the vein over a set time interval, it was possible to measure blood velocity in retinal vessels of various diameters, and to calculate volumetric flow rates in those vessels.

Reduced toxicity of intravitreally injected liposome-encapsulated cytarabine.

Cytarabine has been demonstrated to be a more potent inhibitor of fibroblast proliferation than fluorouracil. It is, however, more toxic to the retina. We evaluated the ocular toxicity of liposome-encapsulated cytarabine in vitrectomized and nonvitrectomized rabbit eyes by ophthalmic and histopathologic examination. In the vitrectomized group, doses of up to 26 micrograms were nontoxic to the retina. In nonvitrectomized eyes, doses of up to 55 micrograms were nontoxic. Doses of 85 micrograms resulted in loss of ganglion cells and disorganization of photoreceptor cells. The results obtained in this study demonstrated a substantial reduction in ocular toxicity of liposome-encapsulated cytarabine, as compared with a previous study which employed free drug. By virtue of its reduced toxicity, cytarabine in a liposomal vehicle may be of value in the treatment of ocular proliferative disorders.

Feasibility of blood flow measurement by externally controlled dye delivery.

We are developing a new method of delivering substances locally and repeatedly in the retinal vasculature under external control. This delivery system is based on encapsulating the substance in heat-sensitive lipsomes, which are injected intravenously and lysed by a heat pulse delivered by a laser. The feasibility of using this system with dyes and creating a sharp dye front was tested in vitro and in vivo. The results indicate that the background fluorescence of intact liposomes is minimal but in contrast a dramatic increase in fluorescence is achieved where the dye is released. In vivo tests indicated that only the selected vascular branch fluoresced. Moreover, a sharp dye front could be obtained repeatedly and preserved over significant distances. The presence of a sharp dye front allowed measurements, in vitro, of blood velocity which correlated well (r = 0.985, P less than 0.001) with the average blood velocity values calculated from the known flow rate.

Repetitive, selective angiography of individual vessels of the retina.

Recent experiments have shown the feasibility of using laser or microwave energy for the externally controlled release of both dyes and drugs from temperature-sensitive liposomes (lipid vesicles). In the present study, calcein, a fluorescent dye, was used to improve the in vivo capabilities of this controlled release system. In vitro studies showed that the encapsulation efficiency was improved when compared to carboxyfluorescein, the fluorescent dye used previously. The improved liposome and dye system was used to obtain repetitive, "selective angiograms" in isolated segments of the retina in rabbits and in a nonhuman primate (Macaca mulatta) over a period of up to 2 hours after a single liposome injection. Conventional fluorescein angiography of the primate retina one week after the end of the experiments showed no visible damage, even after more than 400 individual laser exposures.