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Reduced Toxicity of Liposome-Associafed Amphotericin B Injected Intravifreally in Rabbits Claude Tremblay,*t Michael Barza,*^: Francis Szoka,§:j: Moshe Lahav,||H and Jules The ocular toxicity of liposome-intercalated amphotericin B and commerical amphotericin B were compared after intravitreal injection in healthy pigmented rabbits. Ophthalmoscopic observations over 5 weeks following a single intravitreal injection showed vitreal band formation and focal retinal damage after doses of .commercial amphotericin B as low as 5 Mg- Such lesions were not seen in animals given liposomal amphotericin B in doses up to 20 ng. Histopathologic examination showed areas of retinal atrophy or necrosis in five of 16 rabbits given commercial amphotericin B in doses of 5-20 Mg but in none of 16 rabbits given the same doses of liposomal amphotericin B (P = 0.02). Small white vitreal bodies were seen clinically in virtually all animals given liposomal amphotericin B or "empty" (drug-free) liposomes but in only a few animals given commercial amphotericin B; these deposits may represent residual lipid. Concentrations of amphotericin B ranged from 0.4 to 1.0 jug per ml of vitreous humor 5 weeks after injection of 5-20 Mg of either formulation. These studies indicate that liposome association markedly reduces the ocular toxicity of amphotericin B. Invest Ophthalmol Vis Sci 26:711-718, 1985 Amphotericin B is a drug of choice for a number of serious fungal infections of the eye including Candida endophthalmitis, postsurgical fungal endophthalmitis, and mucormycosis, and is an alternative to natamycin in the treatment of fungal keratitis.1"3 Unfortunately, intraocular penetration of amphotericin B is poor following systemic administration in rabbits,4 although modest levels were found in the aqueous and vitreous humor of two patients whose eyes were enucleated because of severe fungal infection. 5 Subconjunctival injection of amphotericin B penetrates the ocular humors of rabbits very poorly4 and is prone to produce severe local tissue reactions.6 Direct intravitreal injection of amphotericin B is attractive because it produces high concentrations at the site of infection in fungal endophthalmitis. Axelrod et al7 found that normal rabbits tolerated intravitreal injections of 10 /u.g of amphotericin B without toxicity if the drug was administered slowly into the center of the vitreous humor. In contrast, Souri and Green1 reported marked retinal damage in this same species with intravitreal doses as low as 1 jig. In one patient with Candida albicans endophthalmitis, intravitreal injection of 5 /xg of amphotericin B appeared to produce no residual ocular toxicity.8 Recent studies have indicated that incorporation of amphotericin B into liposomes markedly reduces the acute toxicity of the drug while retaining efficacy in animal models of fungal infection.9"" We have developed a liposomal formulation of amphotericin B, which is approximately one-fifth as toxic as the commercial drug in terms of acute lethality after intravenous injection in mice but is fully active in vitro and in the treatment of systemic candidiasis in mice." a In the present study we have compared the ocular toxicity of commercial amphotericin B with that of our liposomal formulation after intravitreal injection in rabbits. Materials and Methods Drug Preparations From the Infectious Disease Division, Department of Medicine,* and the Department of Ophthalmology, || Tufts-New England Medical Center, Boston, Massachusetts, and the School of Pharmacy^ University of California, California. Supported in part by research grant EY-01517 from the National Eye Institute,:): the Massachusetts Lions Eye Research Fund, Inc.,H and Fonds F.C.A.C. pour l'Aide et le Soutien de la Recherche,f Province of Quebec, Canada. Submitted for publication: September 30, 1983. Reprint requests: Michael Barza, MD, New England Medical Center, 171 Harrison Avenue, Boston, MA 02111. Amphotericin B was obtained from the Squibb Institute (Princeton, NJ) as the commercial preparation for intravenous use. This formulation contains sodium desoxycholate as a solubilizing agent. Small unilamellar vesicles were prepared by sonication in a bath type sonicator 12 from a lipid composition of egg phosphatidylcholine (Avanti Polar Lipids; Birmingham, AL) cholesterol, and tocopherol succinate (Sigma Chemical; St. Louis, MO) in a 711 Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 712 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Moy 1985 molar ratio of 5:3:1. The lipids were deposited on the sides of a round bottom flask by evaporation from a chloroform solution. Amphotericin B in dimethylsulfoxide was added to the dried lipids at a 10 mole percent ratio. Phosphate-buffered saline (PBS; composition 137 mM NaCl, 2.6 mM KC1, 6.4 mM Na 2 HPO 4 , pH 7.4) was added to the flask to rehydrate the lipids and to give a final lipid concentration of 50 jumol/ml. The dispersion was hydrated under nitrogen for 10 min and then sonicated under nitrogen for 60 min at room temperature. The slightly opalescent yellow preparation was dialyzed at 4°C against 200 volumes of PBS, changed two times over a 24-hr period to remove nonintercalated amphotericin B and dimethylsulfoxide. A typical preparation retained 70% of the initial amphotericin B (3525 ng amphotericin B per 50 /xmoles total lipid per ml). The liposomes were sterilized by filtration through a sterile 0.22 jum filter (Millipore; Bedford, MA) into a sterile container. Drug concentration in the liposome dispersion was determined by diluting an aliquot of the final preparation 1/1000 in methanol, measuring the absorbance at 388 nm and 406 nm, and comparing it with a standard curve prepared from solid amphotericin diluted in methanol. The standard curve was linear up to 6 Mg/ml of amphotericin B. The liposome weight average diameter was estimated by dynamic light scattering using a Malvern light scattering monochromator (Cherry Hill, NJ) coupled to a Spectraphysics 15 mW helium (633 nm) fixed wavelength laser. Injections Twenty healthy pigmented (Dutch-belted) rabbits, weighing 1.5-2.5 kg, were used. All studies were carried out in accordance with the ARVO Resolution on the Use of Animals in Research. Amphotericin B and liposomal amphotericin B were diluted in sterile distilled water to an appropriate concentration such that the intravitreal dose would be contained in 0.1 ml. The concentration of drug in the liposomal suspension was verified by spectrophotometric absorbance at 388 nm and 406 nm. Each of the forty eyes was randomly assigned to receive one of 10 intravitreal regimens: amphotericin B or liposomal amphotericin B in a dosage of 1, 5, 10, or 20 ng, or a control preparation consisting of PBS or drug-free ("empty") liposomes. The latter were prepared in the same way as drug-containing liposomes. The controls given empty liposomes received the same amount of lipid as was present in the 20 fig injection of liposomal amphotericin B. Each preparation was administered to four eyes in Vol. 26 four animals. The volume injected was 0.1 ml in each instance. In preparation for the intravitreal injections, the rabbits were tranquilized with an intramuscular injection of ketamine 90 mg and acepromazine maleate 1 mg. Proparacaine hydrochloride 0.5% was then applied topically. The intraocular volume was decreased by aspiration of 0.1-0.2 ml of aqueous humor through a 25-gauge needle. The superior rectus muscle was gently grasped with a forceps to stabilize and proptose.the globe. A 27-gauge needle attached to a tuberculin syringe was introduced about 5 mm posterior to the limbus and was inserted to the approximate center of the vitreous humor. The solution (0.1 ml) was injected slowly. Ophthalmoscopic and Histologic Studies The rabbits' eyes were examined once a week with a direct ophthalmoscope and a slit lamp after dilatation with atropine sulfate 1%. All examinations were made by one observer who did not know which preparation had been administered. After 5 weeks, the animals were killed with an intravenous injection of pentobarbital, and the eyes were promptly enucleated. The vitreal lesions appeared stable by this time. About 0.4 ml of vitreous humor was aspirated through a 25-gauge needle for assay of the amount of amphotericin B remaining (see below); this volume was promptly replaced by an equal volume of phosphate-buffered saline to maintain the volume of the globe. The eyes were placed in 10% phosphate-buffered formalin. After fixation, they were properly oriented and a pupil-optic nerve section was cut along the vertical axis. A central segment was processed for paraffin embedding, sectioned at 6 /xm, and stained with hematoxylin and eosin. Selected sections were stained with periodic acid-Schiff reagent. Sequential sections were observed for inflammatory changes in the vitreous humor and for retinal abnormalities. All interpretations were made by one observer who was not aware of which preparation had been administered. The changes were graded as follows: Vitreal inflammation: Vitreal inflammation was graded as follows: ± = few monocular cells along vitreous base per high power field (40X on AO Ultrastar microscope, American Optical USA; Buffalo, NY); 1+ = 10-20 cells along the vitreous base per high power field; 2+ = 20-40 cells per high power field at the vitreous base and elsewhere; 3+ = >40 cells per high power field at the vitreous base and elsewhere. Retinal atrophy or necrosis: These changes were designated as focal or diffuse as estimated by the Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 713 TOXICITY OF INTRAVITREAL LIPOSOAAAL AMPHOTERICIN D / Trembloy er ol. No. 5 anteroposterior involvement in sections from the central segment of the globe. VITREAL BODIES Assay of Amphotericin B in Vitreous Humor Residual amphotericin B was assayed by an HPLC method followed methanol extraction, with o-nitrophenol serving as internal standard.13 Activity was read spectrophotometrically at 388 nm. 0 1+ None 5-10 per eye None Minimal 2+ 10-25 per eye Moderate 3+ >25 per eye Severe l "" 0 Retinal damage None Silvery sheen or atrophy of retina occupying 0.5-1 whole field visible at one time Retinal sheen or atrophy occupying > 1 field visible at one time More extensive than above (no such lesions actually found) LESION Lip-AMB Sug AMB S ug 2+ 1+ During the first week after injection, all but four eyes showed a moderate diffuse inflammatory cellular reaction in the vitreous humor, particularly in the anterior part, and all but six eyes showed scattered small white vitreal bodies. By the second and third weeks, the diffuse inflammatory reaction decreased or disappeared although the vitreal bodies remained. These opacities were sparkling white, were less than 1 mm in diameter, were mostly located in the lower part of the eye, and did not move when the animal's head was moved. In some eyes, bands of "veil-like" vitreal condensations began to appear, sometimes seeming to exert traction on the retina. During the fourth and fifth weeks, the vitreal bodies and vitreal bands remained unchanged. Retinal damage was noted in a few eyes, being manifested by a silvery sheen of the retina, generally in areas adjacent to the fibrotic vitreal bands. In a few animals, slitlamp examination revealed cataract formation. A grading system, summarized in Table 1, was used to quantify the extent of these gross changes 5 weeks after injection when vitreal lesions appeared to be stable. Intermediate values were scored with halfTable 1. Scoring system for vitreal and retinal abnormalities observed on direct ophthalmoscopy Vitreal bands 3+ OF Ophthalmoscopic Examination Vitreal opacities J J«»-r GRADE Results Grade AMB 1ug 1 11 44 3+ Lip-AMB 10 ug 11 Lip-AMB 20 ug J LULI Empty liposomes 1 2 3 EYE NUMBER 4 1 2 3 4 EYE NUMBER Fig. 1. Gross toxicity of intravitreal amphotericin B. Extent of vitreal opacities observed ophthalmoscopically 5 weeks after intravitreal injection of commercial amphotericin B (AMB) or liposomal amphotericin B (lip-AMB) in the indicated dosages in pigmented rabbits. Each bar is the result in one eye using the scoring system shown in Table 1. Control eyes received phosphate-buffered saline (PBS) or empty (drug-free) liposomes. increments (eg, between 1+ and 2+ = 1.5). Figure 1 shows the density of vitreal bodies for each of the four eyes in the eight treatment groups and two control groups (PBS and drug-free or "empty" liposomes). As can be seen, vitreal bodies were evident in most animals given liposomal amphotericin B or empty liposomes. It is not clear that these opacities signify a toxic reaction; they may, instead, represent residual liposomes. However, some were seen in eyes that received no liposomes. Figure 2 shows the extent of vitreal band formation and of retinal damage seen clinically in these same animals. These reactions, which were clearly due to adverse drug effects, were evident in most eyes given amphotericin B in doses of 5 /xg or more, but were absent from all but one of the eyes treated with liposomal amphotericin B. Cataracts were observed in four eyes, one each treated with amphotericin B, 5 jug, 10 /zg, or 20 /ug and one given liposomal amphotericin B, 5 ug. A faint focal cortical opacity was seen in one eye treated Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 714 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / May 1985 |_| VITREAL BAND FORMATION | RETINAL DAMAGE 4+ 3+ 2+ 14- AMB 1 ug • I H 0 44 - 24 • i 1+ OF 0 LESION Lip-AMB Sug AMB 5ug 3+ GRADE Lip-AMB 1ug r-m P | 4+ 3+ Lip-AMB 10 ug AMB 10 ug 2+ 1+ o • IIIL B• H• 1 4+ 3+ 2+ AMB 20 ug Lip-AMB 20 ug 1+ 0 _ 'Ill | 44 34 24 Empty liposomet PBS 14 o 1 2 3 4 EYE NUMBER EYE NUMBER Fig. 2. Gross toxicity of intravitreal amphotencin B. Extent of vitreal band formation and retinal damage observed ophthalmoscopically 5 weeks after intravitreal injection of commercial amphotericin B (AMB) or Hposomal amphotericin B (lip-AMB) in the indicated dosage in pigmented rabbits. Each bar is the result in one eye using the scoring system shown in Table I. Control eyes received phosphate-buffered saline (PBS) or empty (drug-free) liposomes. with liposomal amphotericin B, 10 ug. The lenticular opacities were located in the posterior cortex. Histologic Studies The results of histologic examination are summarized in Table 2. Vitreal inflammation, manifested primarily by round cells along the vitreous base, was Vol. 26 generally slight. Although some inflammation was noted in all groups, there was a suggestion of a somewhat more marked reaction in animals receiving higher doses of liposomal amphotericin B. We did not analyze vitreal band formation histologically because of the possibility that some of the effects observed may have been caused by aspiration of the vitreous humor for assay of amphotericin B. Microscopic examination of the retina revealed damage in five eyes, all of which had been treated with amphotericin B. In one eye injected with amphotericin B, 5 ug, there was atrophy and necrosis over 80% of the inferior retina. Another eye treated in the same manner exhibited focal (2 X 1 mm) retinal atrophy (Figs. 3A and B). One eye injected with amphotericin B, 10 ug, displayed retinal necrosis in one area inferiorly ( 4 X 4 mm) and hypertrophy of the retinal pigment epithelium. A fourth eye, which had been treated with amphotericin B, 20 ug, showed retinal atrophy and focal atrophy of the retinal pigment epithelium (3 X 1 mm). In the fifth eye, injected with amphotericin B, 20 ixg, diffuse necrosis was seen over the lower half of the retina (Figs. 4A and B). The first three eyes described above had been noted to have retinal damage on ophthalmoscopic examination. Overall, five of 16 eyes treated with amphotericin B, but none of 16 treated with liposomal amphotericin B, exhibited histologic retinal abnormalities (P = 0.0217 by Fisher exact test, twotailed). All of the microscopic abnormalities were observed with doses of amphotericin B of 5 fig or more. None of the eyes injected with "empty" liposomes showed retinal damage histologically. Vitreal Concentrations of Amphotericin B Figure 5 shows the concentrations of amphotericin B measured in the vitreous humor 5 weeks after injection. Several specimens were lost for technical reasons. No drug was detectable in eyes given 1 ug of amphotericin B and an unexpectedly high concen- Table 2. Histologic abnormalities in rabbits given intravitreal amphotericin B (AMB) or liposomal amphotericin B (lip-AMB) (each group included four eyes) Vitreal inflammation Lip-AMB AMB Dose of AMB ± + 0 (empty liposomes) I 5 10 20 1 3 2 1 Total ++ ± 1 1 1 2 2 1 2 8 4 0 5 + Retinal abnormalities ++ AMB Lip-AMB 1 2 1 3 1 0 0 2 (1 focal, 1 diffuse) 1 (focal) 2 (1 focal, 1 diffuse) 0 0 0 0 0 7 1 Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 No. 5 TOXICITY OF INTRAVITREAL UPOSOMAL AMPHOTEPJCIN D / Trembloy er ol. 715 Fig. 3. A, top. Equatorial retinal lesion in a rabbit injected intravitreally with 5 /ig of amphotericin B. Note atrophy of all layers, more marked in the inner layers of the retina. The outer nuclear layer is thinned (hematoxylin and eosin, XI20). B, bottom. Equatorial retina in a rabbit injected with 5 fig of liposomal amphotericin B. The retinal architecture is normal (hematoxylin and eosin, XI20). tration of 1.4 ^g/ml was detected in the only eye studied after 1 jug of liposomal amphotericin B. Concentrations were in the range of 0.4-1.0 jug/ml in eyes injected with 5-20 ng of amphotericin B or liposomal amphotericin B. There was no obvious difference in concentration between eyes treated with liposomal or nonliposomal preparations. If one considers that, in these small rabbits, the initial dose of Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 No. 5 TOXICITY OF INTRAVITREAL LIPOSOMAL AMPHOTERICIN D / Trembloy er ol. 715 '•I 3 ,; Fig. 3. A, top. Equatorial retinal lesion in a rabbit injected intravitreally with 5 fig of amphotericin B. Note atrophy of all layers, more marked in the inner layers of the retina. The outer nuclear layer is thinned (hematoxylin and eosin, XI20). B, bottom, Equatorial retina in a rabbit injected with S jig of liposomal amphotericin B. The retinal architecture is normal (hematoxylin and eosin, XI20). tration of 1.4 fig/ml was detected in the only eye studied after 1 fig of liposomal amphotericin B. Concentrations were in the range of 0.4-1.0 fig/ml in eyes injected with 5-20 fig of amphotericin B or liposomal amphotericin B. There was no obvious difference in concentration between eyes treated with liposomal or nonliposomal preparations. If one considers that, in these small rabbits, the initial dose of Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 No. 5 717 TOXICITY OF INTRAVITREAL UPOSOMAL AMPHOTERICIN D / Trembloy er ol. four half-lives, suggesting a vitreal half-life of amphotericin B of approximately 9-12 days. Discussion Amphotericin B has been a mainstay in the treatment of fungal endophthalmitis. 14~16 Jones2 has documented that within the past two decades, candidal endophthalmitis has emerged as the most common ocular fungal infection. Several case reports support the effectiveness of amphotericin B, given by subconjunctival 1718 or intravitreal8 injection in the treatment of fungal endophthalmitis. However, the toxicity of amphotericin B may limit its effectiveness. Recent experiments in our laboratory have shown that liposome association of amphotericin B strikingly reduces the toxicity of the drug after systemic administration in mice. The acute LD 50 of commercial amphotericin B in this animal is approximately 2.27 mg/kg, whereas the acute LD 50 of the liposomal preparation is 11.75 mg/kg. lla Similar reductions in toxicity with liposomal formulations of amphotericin B have been reported by others. 9 "" We have also shown that liposomal amphotericin B retains full antifungal activity in vitro.1 la Finally, we" a and others 9 "" have demonstrated that liposomal amphotericin B can be given safely in higher doses than commercial amphotericin B to animals with systemic fungal infections thereby achieving higher survival rates. The small unilamellar vesicles that we used have a diameter of approximately 0.1 ^m so that they can be sterilized by passage through a 0.22 j^m filter. The reduced systemic toxicity of liposomal amphotericin B led us to test the preparation for ocular toxicity after intravitreal injection in rabbits. During the first 1 to 2 weeks after intravitreal injection of amphotericin B or liposomal amphotericin B, a vitreal haze consistent with leukocytic infiltration was observed in most eyes. Thereafter, the major clinical changes consisted of vitreal "bodies", ie, small white sparkling opacities mainly located in the lower part of the eye, vitreal condensations, and retinal abnormalities. Vitreal bodies were noted predominantly in animals given liposomal preparations. They may represent residual lipid. However, they were also noted in some eyes treated with nonliposomal formulations. Their histologic counterparts may have been eluted during tissue processing. Further studies will be needed to elucidate the nature of these particles and to determine how long they remain in the eye. Vitreal band formation and retinal damage of grade 1+ or greater were clinically evident only in eyes treated with amphotericin B alone. This strongly suggests that liposome association reduces the acute inflammatory effect of the drug. Cataract formation was observed in one of 20 eyes given amphotericin __ 2 . 0 - 1 1.8m .E 1.4- '| 1.2- o o 1.60 o o f 1.0o = 0.6- • o o o • ° 1 o . • § Lip-AMB AMB 0.4- lo.2«5 o AMB Lip-AMB ug AMB Lip-AMB 5 ug AMB 10 ug Lip-AMB 20 ug Fig. 5. Concentration of amphotericin B in the vitreous humor of rabbits 5 weeks after intravitreal injection of commercial amphotericin B or liposomal amphotericin B in the dosage indicated. Each point is the result for one eye. Assays were done by HPLC. B and in three of 20 eyes given liposomal amphotericin B. Although we cannot exclude trauma as a cause of the cataracts, we were careful to direct the needle posteriorly. Neither the distribution of cataracts among the treatment groups nor their morphologic appearance permits us to interpret their cause or significance. On histologic examination, vitreal inflammation, chiefly in the form of a round-cell infiltrate along the vitreous base, was noted in over half of the eyes, including two eyes treated with PBS. Inflammation of grade 1+ or 2+ was noted in four of 16 eyes injected with amphotericin B but eight of 16 injected with liposomal amphotericin B, suggesting a somewhat more irritating effect of the liposomal drug. However, the relation between vitreal inflammation and substance or dose of substance administered was inconclusive. If we consider retinal necrosis or atrophy as the most significant histologic lesions, there was a marked benefit of liposomal amphotericin B. Retinal damage was detected histopathologically in five of 16 eyes treated with amphotericin B but none of 16 given liposomal amphotericin B (P = 0.0217 by Fisher exact test). This corresponds to the clinical finding that significant retinal damage was confined to the animals given amphotericin B alone. Although it is conceivable that needle trauma caused the atrophic changes noted in two of the eyes, it is unlikely to have produced the more extensive changes observed in the other three eyes. Moreover, only animals given nonliposomal drug manifested these abnormalities, which should not be the case if trauma due to the injection were responsible for the changes. We did not control for the direction of the needle bevel in making the injections, but we presume that this varible was randomly distributed among the eyes. We cannot rule out the possibility that the desoxycholate solu- Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017 718 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1985 bilizer present in commercial amphotericin B played some role; however, Axelrod et al7 found that desoxycholate was not toxic in the doses used in this study. The mechanism by which liposome-association reduces the toxicity of amphotericin B without reducing its antifungal activity is not clear. In contrast to water-soluble molecules, which are trapped in the interior (aqueous phase) of liposomes, amphotericin B is presumably intercalated in the wall of the liposome.19 The antibiotic has an affinity for sterols present in the liposomes and in human and fungal cellular membranes. Its greatest affinity is for ergosterol, which occurs naturally in fungal cells but not human cell membranes. It may be that the interaction between amphotericin B and human cell membranes is reduced by the competitive effect of the liposomal cholesterol but that the drug is readily captured by fungal ergosterol. Concentrations of amphotericin B of 0.4-1.0 /xg/ ml were found in the vitreous humor 5 weeks after a single intravitreal injection of 5-20 ng. The apparent clearance rate of amphotericin B from the vitreous humor was similar for liposomal and nonliposomal preparations. However, our data do not permit us to determine whether amphotericin B administered in liposomes was cleared as the drug-liposome complex or after the drug had become separated from the liposomes. More detailed pharmacokinetic studies underway in our laboratory suggest that amphotericin B has a long half-life in the vitreous humor, choroid, and retina following intravitreal injection. Accordingly, intravitreal injections may not have to be repeated often to maintain relatively high levels of drug in pertinent sites. The results of this study offer some hope that liposome-intercalation may reduce the toxicity of intravitreal amphotericin B, thereby permitting this valuable drug to be used more safely in the treatment of fungal endophthalmitis. Our data suggest that further study of the safety and efficacy of intraocular liposomal amphotericin B is warranted. Key words: amphotericin B, liposomes, intravitreal, toxicity, rabbit References 1. Souri EN and Green WR: Intravitreal amphotericin B toxicity. Am J Ophthalmol 78:77, 1974. Vol. 26 2. Jones DB: Therapy of postsurgical fungal endophthalmitis. Trans Am Acad Ophthalmol Otolaryngol 85:357, 1978. 3. Ghosheh R: Polyene antibiotics. In Antimicrobial Agents in Ophthalmology, Smolin G and Okumoto M, editors. New York, Masson Publishing USA, 1983, pp. 107-110. 4. Green WR, Bennett JE, and Goos RD: Ocular penetration of amphotericin B: a report of laboratory studies and a case report of postsurgical cephalosporium endophthalmitis. Arch Ophthalmol 73:769, 1965. 5. Fisher JF, Taylor AT, Clark J, and Rao R: Penetration of amphotericin B into the human eye. J Infect Dis 147:164, 1983. 6. Bell RW and Ritchey JP: Subconjunctival nodules after amphotericin B injection. Arch Ophthalmol 90:401, 1973. 7. Axelrod AJ, Peyman GA, and Apple DJ: Toxicity of intravitreal injection of amphotericin B. Am J Ophthalmol 76:578, 1973. 8. Stern GA, Fetkenhour CL, and O'Grady RB: Intravitreal amphotericin B treatment of Candida endophthalmitis. Arch Ophthalmol 95:89, 1977. 9. Taylor RL, Williams DM, Craven PC, Graybill JR, Drutz DJ, and Magee WE: Amphotericin B in liposomes: a novel therapy for histoplasmosis. Am Rev Respir Dis 125:610, 1982. 10. Graybill JR, Craven PC, Taylor RL, Williams DM, and Magee WE: Treatment of murine cryptococcosis with liposome-associated amphotericin B. J Infect Dis 145:748, 1982. 11. Lopez-Berestein G, Mehta R, Hopfer RL, Mills K, Kasis L, Menta K, Fainstein V, Luna M, Hersh EM, and Juliano R: Treatment and prophylaxis of disseminated infection due to Candida albicans in mice with liposome-encapsulated amphotericin B. J Infect Dis 147:939, 1983. lla.Tremblay C, Barza M, Fiore C, and Szoka F: Efficacy of liposome-intercalated amphotericin B in the treatment of systemic candidiasis in mice. Antimicrob Agents Chemother 26: 170, 1984. 12. Szoka F and Papahadjopoulos D: Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng 9:467, 1980. 13. Mayhew JW, Fiore C, Murray T, and Barza M: An internallystandardized assay for amphotericin B in tissues and plasma. JChromatogr 274:271, 1983. 14. Michelson PE, Stark W, Reeser F, and Green WR: Endogenous Candida endophthalmitis. Int Ophthalmol Clin 2:125, 1971. 15. Edwards JE Jr, Foos RY, Montgomerie JZ, and Guz LB: Ocular manifestation of Candida septicemia: review of 76 cases of hematogenous Candida endophthalmitis. Medicine 53:47, 1974. 16. Clarkson JG and Green WR: Endogenous fungal endophthalmitis. In Clinical Ophthalmology, Duane TD, editor. Hagerstown, Harper and Row, 1976, pp. 1-44. 17. Allen HF: Amphotericin B and exogenous mycotic endophthalmitis after cataract extraction. Arch Ophthalmol 88:640, 1972. 18. Glassman MI, Henkind P, and Alture-Werber E: Monosporium apiospermum endophthalmitis. Am J Ophthalmol 76:821, 1973. 19. Andreoli TE: On the anatomy of amphotericin B-cholesterol pores in lipid bilayer membranes. Kidney Int 4:337, 1973. Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933354/ on 05/12/2017