BritishJ7ournal ofOphthalmology 1994; 78: 677-680

Treatment of cytomegalovirus retinitis withintravitreal injection of liposome encapsulatedganciclovir in a patient with AIDS

Shiva K Akula, Patrick E Ma, Gholam A Peyman, Mohamad H Rahimy,Newton E Hyslop Jr, Aileen Janney, Paul Ashton

Department of Medicine,Tulane UniversityMedical Center, NewOrleans, USAS K AkulaN E Hyslop

LSU Eye Center, NewOrleans, USAPEMaG A PeymanM H Rahimy

Department ofMicrobiology, MedicalCenter of Louisiana atNew Orleans, USAA Janney

Department ofOphthalmology,University of Kentuckyat Lexington, USAP AshtonCorrespondence to:Dr Gholam A Peyman, LSUEye Center, 2020 GravierStreet, Suite B, New Orleans,LA 70112-2234, USA.Accepted for publication11 April 1994

Figure I Right eye oninitial visit beforeintravitreal ganciclovir.

AbstractTo study its safety and efficacy in treatingcytomegalovirus (CMV) retinitis, an AIDSpatient received an intravitreal injection ofliposome encapsulated ganciclovir in the righteye. The left eye served as a control, receivingintravitreal free ganciclovir. The right eyeshowed no retinal haemorrhages or detach-ment; however, vision declined initially,stabilising later. Weekly examination showedneither progression of the CMV retinitis nornew lesions in the right eye. The left eyeshowed reactivation of old CMV retinitis.Liposome encapsulated ganciclovir reducedthe number of intravitreal injections, stabilis-ing CMV retinitis, and warrants further study.(BrJ Ophthalmol 1994; 78: 677-680)

Cytomegalovirus (CMV) retinitis is the mostcommon cause of visual loss in AIDS. Approx-imately 30% of patients with AIDS are expectedto develop CMV retinitis at some time duringtheir illness.' Ganciclovir (Cytovene, SyntexLaboratories Inc, Palo Alto, CA, USA) has beenavailable since 1989 in intravenous form.Because intravenous ganciclovir causesmyelosuppression and thrombocytopenia, thuspredisposing to line cannula sepsis and prevent-ing concomitant use of zidovudine, intravitrealganciclovir was given. The half life ofintravitrealganciclovir has been estimated at 13-3 hours2;therefore, induction therapy with intravitrealganciclovir generally consists of two to three

injections per week for 2 to 3 weeks. Main-tenance therapy consists of lifetime weekly injec-tions. Repeated intravitreal injections increasethe risk of endophthalmitis, damage to lens, andretinal detachment, and may be poorly toleratedby end stage patients. To prevent theseproblems, ganciclovir was encapsulated inliposomes to increase the intravitreal retention ofthe drug, thereby decreasing the frequency ofinjections.

Case reportA 32-year-old white man, HIV positive since1987, had a clinical diagnosis ofCMV retinitis inAugust 1990 at ophthalmic evaluation forblurred vision (Fig 1). Initial visual acuity was20/20 -2 in the right eye and 20/25 +2 in the lefteye. Zone III lesions and zone I/II lesions werenoted in the right and left eyes, respectively.Intravenous ganciclovir therapy at 300 mg twicedaily was initiated but later complicated byneutropenia and Staphylococcus aureus endo-carditis. After adequate treatment for both thesecomplications, intravenous ganciclovir wasrestarted. Subsequent interruption of intra-venous ganciclovir for recurrent neutropenia andcannula sepsis prompted removal of the cannulaand the use of intravitreal ganciclovir. On 18September 1991, twice weekly 200 [ig doses ofintravitreal ganciclovir were started in the righteye. Vision in the right eye was 20/25 +3 and inthe left eye was counting fingers temporal vision.On 27 September 1991, intravitreal ganciclovirwas increased to 400 [tg and maintained at 400 [igweekly in the right eye. On 12 October 1991,vision in the right eye decreased to 20/400 oneday after an intravitreal ganciclovir injection.The patient noticed pain, photophobia in theright eye, and endophthalmitis was diagnosed.He was admitted for vitreal needle biopsy andintravitreal ganciclovir injection was given alongwith intravitreal gentamicin and clindamycin.Subsequently, the vitreous cultures grewStaphylococcus epidermidis and intravitrealvancomycin and clindamycin were reinjected.The intravitreal injections were stopped andintravenous ganciclovir started with a visualacuity improvement to 20/400 with moderatevitreous haze.

In light of the recurrent neutropenia, severalepisodes of cannula sepsis, and peripheral intra-venous access intolerance the patient was givenan intravitreal injection ofliposome encapsulatedganciclovir in the right eye on 10 February 1992(Figs 2 and 3). Liposome encapsulation was usedfor slow release in the vitreous. Before the

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liposome encapsulated ganciclovir injection, thepatient's informed consent was obtained, alongwith the approval of human subjects committeeat Tulane University School of Medicine. Theleft eye was given regular intravitreal ganciclovirtwice weekly. Three days later, visual acuity wasreduced to hand movements and 2+ vitreoushaze was noted in the right eye. Vitreal taps oftheright eye were done 7, 14, and 24 days after thesingle intravitreal liposome encapsulated ganci-clovir injection. Follow up of the patient wasinterrupted because of multiple opportunisticinfections, including Mycobacterium avium intra-cellular complex and disseminated Kaposi'ssarcoma, leading to worsening physical condi-tion. The family and the patient decided todiscontinue further intravitreal therapy, andvision deteriorated to bilateral blindness. Thepatient died on 21 June 1992.

Materials and methodsA reverse phase evaporation method of liposome

preparation was used for encapsulation of ganci-clovir. The lipid composition for this formula-tion was a 4:1 molar ratio ofphosphatidylcholineand phosphatidylglycerol from Avanti PolarLipids, Inc (Alabaster, AL, USA). The lipidswere dissolved in an organic solvent (ether andchloroform, 2:4, v:v). The organic phase wasthen combined with an aqueous phase containingthe drug ganciclovir to be encapsulated. Theorganic phase was three times the aqueous phaseinitially. The two phase mixture was then soni-cated for 5 minutes to form a milky, homoge-neous emulsion. This emulsion containedreverse micelles and was evaporated to removethe organic solvent under vacuum by a rotaryevaporator. This method allows for preparationof large unilamellar vesicles with relatively highencapsulation efficiency. After completion ofliposome formation, the encapsulated drug wasseparated from unencapsulated material by sub-jecting the preparation to dialysis for 24 hours.

STERILISATIONAll equipment and glassware were sterilisedbefore liposome drug preparation. Furthermore,after liposome formulation, the liposome ganci-clovir preparation was filtered using a 0 2 [imfilter,and an aliquot was placed on blood agar andbrain heart infusion medium to test for possiblecontamination. No bacteria or fungi weredetected by culture.

SAMPLE COLLECTIONThe right eye was irrigated with sterile salinesolution. Retrobulbar anaesthesia was used. Acotton pledget was applied to the cornea scleralsurface with topical anaesthesia. Vitreal sampleswere collected with a No 30 1/2 inch tuberculinsyringe. A 200 tl sample of vitreous wasremoved at 0 (baseline), 7, 14, and 24 days after asingle intravitreal injection of 0-25 ml (1 mg) ofthe liposome encapsulated ganciclovir. Thesamples were immediately stored frozen at-20°C until ganciclovir analysis.

GANCICLOVIR ASSAYThe vitreous samples were analysed for ganci-clovir by the high performance liquid chroma-tography (HPLC) method using a 25 cmx5 ,umC18 reverse phase column. The mobile phase was2% acetonitrile/ionised water (v/v) and 0-02%acetic acid; pH 4 0 with a flow rate of 1 ml/min.The detection wavelength was 254 nm. Underthese conditions, the retention time of ganci-clovir was 7-8 minutes and the detection limit0-02 tig/l.

ResultsOphthalmoscopic examination of the right eyeshowed no progression of old CMV retinitis andno new lesions were noted. There was noevidence ofretinal haemorrhages or detachment,but vision declined initially owing to vitreousclouding. Because the visual acuity is not alwayseasy to determine in patients with poor overallcondition,3 it was not used as a criterion for

Figure 2 Right eye I hourbefore the intravitrealinjection of liposomeencapsulated ganciclovir.

Figure 3 Right eye I dayafter intravitreal injection ofliposome encapsulatedganciclovir.

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Treatment ofcytomegalovirus retinitis with intravitreal injection ofliposome encapsulated ganciclovir in a patient with AIDS

treatment evaluation.4 Furthermore, visualacuity is dependent upon the location of thelesions but not on their activity. The intra-vitreally injected liposome encapsulated ganci-clovir appeared as a whitish suspension located inthe inferior part of the vitreous. The cloudingdisappeared gradually over 2 weeks. The left eyeshowed reactivation ofold CMV in the first week,requiring twice weekly intravitreal injections ofganciclovir.

PHARMACOKINETICS OF LIPOSOME ENCAPSULATEDGANCICLOVIRVitreous ganciclovir concentration was 2-5 ,ug/mlbefore the injection of liposome ganciclovir. Theliposome encapsulated ganciclovir preparationachieved a vitreous drug level of 11 6 [ig/ml at 7days after treatment. Ganciclovir levels remainedat 4-2 and 6-6 [ig/ml 14 and 24 days aftertreatment, respectively (Table 1).

DiscussionCMV retinitis is the most frequent ocular oppor-tunistic infection of the retina in AIDS, withprevalence ranging from 15% to 40%.' BecauseCMV is a multisystem disease, the systemic routeof treatment is often used. Intravenous ganci-clovir causes neutropenia in 16-75% of treatedpatients,"' precluding the concomitant use ofzidovudine, the drug known to prolong survivalin AIDS. Systemic forcarnet91" is nephrotoxic in30% of patients and can cause metabolic abnor-malities of calcium and magnesium. Seizureshave also been reported. Both ganciclovir andfoscarnet require a long term indwelling centralvenous cannula for administration. Althoughsubstantially lower health costs are associatedwith daily home infusion through a centralvenous cannula, the patients have increased riskof cannula related infection, sepsis, or throm-bosis.6 '°The pharmacokinetics of intravenous ganci-

clovir revealed comparable levels in subretinalfluid and in blood (7 16 and 8- 16 mmol/l respec-tively). The subretinal fluid levels decreased to0-8 mmol/l 21 hours after intravenous adminis-tration of the ganciclovir."The advantages of intravenous ganciclovir

therapy include the ability to treat bilateraldisease and possible prevention or control ofCMV infections at other sites. Because the role ofintravenous ganciclovir in survival is unclear andsystemic toxicities are associated with intra-venous ganciclovir and foscarnet, the intravitrealroute of administration is being studied.3 4 12-15 Inrabbit eyes,'6 200-400 pg doses of ganciclovirshowed no evidence of adverse electrophysio-logical or histopathological effect. However,high doses (155 mmol/l) in vitrectomy infusionfluids were associated with significant retinalcellular disorganisation and dissipation. Becausethe half life of intravitreal ganciclovir is 13-3hours,2 frequent administration is required, lead-ing to patient intolerance. Repeated intravitrealganciclovir administration is associated withincreased risk ofendophthalmitis, retinal detach-ment, vitreous haemorrhage, and increasedintraocular pressure.21' 1' The incidence of bac-

terial endophthalmitis varied from zero in 110injections4 to 0 4% in a series of 249.'7 Otherreported complications include conjunctivalscarring and scleral induration.

Because there is no animal model for CMVretinitis, in vitro studies are heavily relied upon.Fifty per cent reduction ofviral plaque formation(minimal inhibitory concentration [MIC50]) and areduction in DNA synthesis occur at a ganci-clovir concentration of 0 5 to 3 0 mmol/l.Another reason for studying intravitreal ganci-clovir is its use in special situations such asjuxtafoveal CMV retinitis lesions. The fovea is arelatively avascular area of the retina, although itis nourished from the choroid.Although 400 pg in 01 ml was used by

Cochereau-Marrin and associates4 withoutserious toxicity, there is inadequate informationto conclude that higher doses show betterefficacy. Doses up to 600 [ig have been usedclinically (personal discussion with Dr P Ma)with no obvious toxicity in controlling foveallesions. The continued use of 400 [tg for initialtherapy was preferred, as it provides intravitreallevels ofdrug higher than the MIC for mostCMVisolates. It is further believed to be effective bymany investigators and will decrease risk of drugtoxicity.Liposomes are membrane-like vesicles used to

encapsulate drugs. They are made from phos-pholipids such as lecithin, phosphatidylglycerol,and phosphatidylserine. Liposomal encapsula-tion before intravitreal injection has been used toenhance a drug's therapeutic index primarily byconfining the drug's action to the local site ofinjection, thereby increasing the amount of drugdelivered to the target tissues; serving as asustained release depot; extending the drug's halflife; and decreasing or minimising intraocularside effects through a controlled release effect.

Experimentally induced streptococcal,staphylococcal, and candida endophthalmitiswere treated with liposome encapsulated peni-cillin,'8 clindamycin," and amphotericin,20respectively, with variable success.

Intraocular drug delivery systems2' haverecently been studied. Ifreasonably longer intra-vitreal retention of ganciclovir is achieved byintravitreal injection, complicated surgicalimplantation of a device can be avoided. Intra-vitreal injection of liposomal ganciclovir allowedthe retention of ganciclovir as long as 24 daysafter injection in a concentration of 6-6 ,ug/ml,which is higher than the MIC50 concentration.The lower concentration of4 2 [tg/ml followed by6-6 [tg/ml may be due to the dilutional effect ofintravitreal sampling.Although subjective deterioration of visual

acuity may be caused by vitreal haze, objectiveretinal examinations revealed no progression orrecurrence of retinitis, suggesting clinical benefitof the sustained release of ganciclovir in thevitreous. The location and size of lesions have a

greater short term effect on visual acuity than thedegree of control by antiviral medications. Theeffect of liposomes on the retina is unclear at thistime. The lack of acute retinal toxicity and theretention of intravitreal ganciclovir 24 days afterthe injection warrant further studies. Higherfractions of liposome encapsulated ganciclovir

Table I Clearance ofanintravitreal injection ofliposome encapsulatedganciclovir in the right eye

Days after Concentrationinjection ([Lg/ml)0 2-57 11-614 4-224 6-6

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may remain longer than 25 days. Althoughprolongation of intravitreal retention of ganci-clovir is important, it needs to'be correlated withimprovement in visual acuity and resolution ofthe CMV retinitis lesions (Table 1).Supported in part by US Public Health Service grants EY07541and EY02377 from the National Eye Institute, National Institutesof Health, Bethesda, MD and GCRC grant 5MOIRRO50 96-05from the NIH.The material in the paper was presented as a poster at the VIII

International Conference on AIDS/Ill STD World Congress inAmsterdam, The Netherlands, 19-24 July 1992.

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