nor-cyclic gmp

Evaluation of Retinal Toxicity and LiposomeEncapsulation of the Anti-CMV Drug 2'-nor-cyclic GMP

Sima Shakiba, Kerry K. Assil, Alan D. Listhaus, David Munguia, Marisa Flores-Aguilar,Chau Vuong, Clayton A. Wiley,* Richard L. Tolman,\ John D. Karkas,^Germaine Bergeron-Lynn, and William R. Freeman

Purpose. Human cytomegalovirus (HCMV) is an important pathogen in the immunocompro-mised patient. CMV retinitis is a leading cause of blindness in patients with AIDS. Ganciclovirand foscarnet are currently the treatments being used for this retinitis, but they both havemajor toxicities when used systemically. Intravitreal therapy with ganciclovir has been used insome patients who cannot tolerate systemic treatment. The major problem with this modality isthe necessity for administration of between 1 and 3 intravitreal injections per eye per week.2'-nor-cyclic GMP is a nucleotide analog, a cyclic phosphate derivative of ganciclovir. Neutralsalts of the compound are extremely water soluble, and the charged phosphate group atneutral pH make it an ideal candidate for encapsulation into a multivesicular liposome system.

Methods. The authors evaluated the retinal toxicity of the diethanolammonium salt 2'-nor-cy-clic GMP by using electroretinographic, morphologic, and ophthalmoscopic techniques afterintravitreal injections in rabbit eye.

Results. The intraocular therapeutic index for 2'-nor-cyclic GMP is 20. At the 10 fig dose,electroretinogram, ophthalmoscopic examination, and both light and electron microscopyrevealed no abnormalities. Toxicity was evident at 50 ng and higher doses with ERG changes(loss of amplitude) and retinal pathology that varied from vacuolization of the retinal pigmentepithelium and loss of height of the outer photoreceptor segment to loss of the entire outerretina. In addition, an in vitro drug release half-life of 1,000 hours (more than 75 times that ofganciclovir) was found for 2'-nor-cyclic GMP in liposome, which may be able to be exploited inthe therapy of patients with CMV retinitis unable to tolerate toxic systemic therapy.

Conclusion. The anti-CMV drug, 2'-nor-cyclic GMP, may be promising for intravitreal injec-tion, particularly if encapsulated into liposomes. Invest Ophthalmol Vis Sci 1993;34:2903-2910.

.Human cytomegalovirus (HCMV) is an importantpathogen in the immunocompromised patient, espe-cially in patients with acquired immunodeficiency syn-drome (AIDS). Retinitis caused by HCMV infection inpatients with AIDS is a leading cause of blindness inthis population. Ganciclovir (DHPG), a hydroxylated

From the Departments of Ophthalmology, *Pathology (Neuropathology), andNeuroscience, University of California, San Diego, California, and f MerckResearch Laboratories, Rahway, New jersey.Supported by N1H EYO 7366, a grant from the University of California AIDS TaskForce, and thejeanette Rubin Family Jewish Community Foundation of San Diego.Submitted for publication August 19, 1992; accepted January 27, 1993.Proprietary interest category: N.f Proprietary interest category: E.Reprint requests: William R. Freeman, M.D., Department of Ophthalmology, ShileyEye Center, University of California/San Diego, 9500 Oilman Drive, La folia,CA 92093-0946.

homolog of acyclovir, is currently the treatment beingused for this retinitis. Licensed for use in the UnitedStates since 1989, ganciclovir is usually successful incontrolling CMV infection, but it is toxic to the bonemarrow and some other organ systems so therapy of-ten must be interrupted. Foscarnet has recently beenlicensed in the United States and has approximatelyequal efficacy as ganciclovir when used systemically,but it also entails major systemic toxicity, particularlyelectrolyte imbalance and nephrotoxicity.1 For thesereasons, local intravitreal therapy has been used in pa-tients who cannot tolerate systemic therapy.23 Intravi-treal therapy with ganciclovir is used in some patientsas 100 to 200 /xg injections on a once-a-week to three-times-a-week basis. The major problem with this mo-dality is the necessity for frequent administration,

Investigative Ophthalmology & Visual Science, September 1993, Vol. 34, No. 10Copyright © Association for Research in Vision and Ophthalmology 2903

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2904 Investigative Ophthalmology & Visual Science, September 1993, Vol. 34, No. 10

which often must be bilateral; thus, patients with bilat-eral CMV retinitis may receive two to six injectionsweekly.4-5-6-7

For this reason, we have been interested in anti-CMV compounds that are polar and that may be incor-porated into the aqueous phase of liposome systems.This would have the advantage of prolonging the in-travitreal half-life of the compound and might allowfor a more clinically achievable administration sched-ule. 2'-nor-cyclic GMP is a nucleotide analog that is thesodium salt of the cyclic phosphate derivative of ganci-clovir.8 The compound is extremely water soluble (100mg/ml); its charged phosphate group would make it anideal candidate for encapsulation into a multivesicularliposome system, which we have used with other com-pounds.91011 The antiviral action of this compound isindependent of thymidine kinase12 and appears tohave a direct activity on viral DNA polymerase. Mostof the antiviral agents (e.g., acyclovir) that have beenused for herpes virus infections depend on their phos-phorylation by the virus-encoded thymidine kinase foractivity. Human CMV, which does not encode for aspecific thymidine kinase, does not show any sensitivityto these antiherpetic drugs. 2'-nor-cyclic GMP is activein vitro against thymidine kinase-deficient herpes sim-plex virus and varicella and those strains that are resis-tant to the "classical" anti-herpes drugs. Of note, gan-ciclovir-resistant CMV strains have been isolated fromimmunocompromised patients treated with ganciclo-vir for CMV infection.13 As suggested by Biron et al,13

this resistance can be based on the impaired phos-phorylation of the drug. It is likely that such strains willbe sensitive to the phosphorylated analogs of ganciclo-vir such as 2'-nor-cyclic GMP, as kinase-dependentherpes resistors were shown to be.14

In animal models of herpes keratitis15 2'-nor-cyclicGMP, appears to be more effective than trifluridine(F3T). It has potent in vitro activity against many DNAviruses, including herpes simplex, HCMV, vaccinia,SV-40, and adenovirus.8 In guinea pig CMV models, ithas a selectivity index (50% cytotoxicity dosage in sta-tionary monolayer guinea pig embryo cells/50% effec-tive dose by plaque reduction assay) of 110, which is10-fold higher than ganciclovir. In plaque reductionassays, 2'-nor-cyclic GMP has approximately 20-foldgreater potency against guinea pig CMV than ganci-clovir.16 In addition, it appears to have a more pro-longed mode of action than does ganciclovir in guineapig CMV tissue culture systems.16 In HCMV (AD 169)models, it is of similar potency to ganciclovir withID50 (drug concentration inhibiting viral plaque by50%) of 2 ^mol/1 compared to 0.43 to 7 jimol/1 forganciclovir.817 Compared to foscarnet, 2'-nor-cyclicGMP has 66 times more potency against HCMV and10 times more potency against HSV-1.813

In summary, 2'-nor-cyclic GMP has several proper-

ties that make it well suited for intraocular delivery.First, the compound produces a long-lasting antiviraleffect that may require a less frequent dosing inter-val.16 Because of its polar nature, the compound maybe encapsulated into liposomes.12 A repository prepa-ration such as liposomes may be advantageous for lo-cal intraocular therapy for CMV retinitis. We there-fore wished to determine the intraocular toxicity andtherapeutic index of 2'-nor-cyclic GMP and its suitabil-ity for encapsulation into a multivesicular liposomesystem. Finally, ganciclovir-resistant CMV strains havebeen isolated from immunocompromised patientstreated with ganciclovir for CMV infection13 and maybe sensitive to 2'-nor-cyclic GMP. For these reasons wechose to evaluate the retinal toxicity of 2'-nor-cyclicGMP in the rabbit eye after intravitreal injection.

MATERIALS AND METHODS

Animal Studies

Twenty-five New Zealand white rabbits, each weighing2.5 to 3 kg, were used. All experimentation was per-formed in accordance with the guidelines of the Asso-ciation for Research in Vision and Ophthalmology.

Electroretinography (ERG) was performed on allrabbits before the procedure and before euthanasia ateither 30 or 60 days as previously described.19 Beforethe procedure, the eyes were dilated with 2 to 3 dropsof 2.5% phenylephrine and 1% mydriacyl and darkadapted for 30 minutes. A mixture of ketamine/xyla-zine was used for anesthesia (21 mg/kg ketamine, 5mg/kg xylazine). The eyes were positioned 6 inchesfrom the Grass stimulator light source. Silver-impreg-nated nylon electrodes were placed on the corneas.Four to eight flash ERGs were averaged, transferredto an amplifier and DA converter by our design, andthen transmitted to a IBM-based personal computer.Data were collected on eyes containing 2'-nor-cyclicGMP or normal saline. Other control data included adata bank of normal ERG amplitudes and latencies inNew Zealand White rabbits corrected for age andweight of the animals.

For intraocular injections, the rabbits were anes-thetized with intramuscular injections of 28 to 56 mg/kg ketamine and 12 mg/kg xylazine. One percent pro-paracaine was used for topical anesthesia of the cor-nea. Under sterile conditions, 0.1 ml of aqueous fromthe anterior chamber was removed with a 27-gaugeneedle, which caused a decrease of the intraocularpressure before drug delivery. The rabbits receivedintravitreal injections of 0.1 ml of 2'-nor-cyclic GMP atdoses of either 10, 20, 50, and 100 ng of drug dis-solved in normal saline, or normal saline. The drug wasinjected with a 25-gauge needle 1 to 2 mm posterior to


Retinal Toxicity of the Anti-CMV Drug 2'-nor-cyclic GMP 2905

the limbus into the vitreous. The tip of the needle wasin the mid-vitreous cavity during the injection.

Each rabbit was examined by indirect ophthalmos-copy before injection, at 5 days after injection whenapplicable, and before euthanasia. At the conclusionof 1, 3, 7, 30, and 60 days, the rabbits were killed andperfusion fixed. All rabbits were deeply anesthetizedwith 80 mg/kg ketamine and 80 mg/kg xylazine mix-ture. After intracardiac injection of 1 ml of heparin1,000 U, an intra-aortic cannula was inserted and thedescending aorta was clamped. Then 1 liter of roomtemperature normal saline was perfused, followed by1 liter of fixative containing 2% paraformaldehydeand 2% glutaraldehyde in 0.1 M Sorenson's phos-phate, pH 7.2. At this time, an incision was made in theright atrium. After the eyes were enucleated, a smallincision was placed at the limbus and the eyes wereplaced for postfixation in the same fixative in a refrig-erator for 1 hour. The samples were then transferredto buffer 0.1 M Sorenson's phosphate, washed threetimes, and refrigerated for gross sectioning. The de-tails of the eye numbers and time points are listed inTable 1.

Histopathologic Methods

Gross sectioning of each eye delivered three sectionsfor histology. After bisecting each eye through the op-tic nerve, one area above and one below the medullaryray were taken from one-half of the globe for electronmicroscopy. A section was removed from the otherhalf of the globe for thin light microscopy (2 /xm sec-tions). All tissues were postfixed in osmium after pre-liminary embedding of each sample in 2.25% agar. Forthin light microscopy, the sections were plastic embed-ded in glycol methacrylate, and the 2-fim thick sectionswere stained with toluidine blue. For electron micros-copy, the sections were embedded in epon-araldite.

Preparation of Liposomes and Drug ReleaseStudiesLiposomes were prepared as previously described.101120

In brief, 7.5 mg of dioleyl phosphatidyl choline, 1.5mg of dimyristoyl glycerol, 1.68 mg of triolein, and5.81 mg of cholesterol were dissolved in 1 ml of chloro-form. One milligram per ml solution of 2'-nor-cyclicGMP diethanolammonium salt was dissolved in 200fimo\/l of sucrose at pH 6.8. One milliliter of this solu-tion was added drop-wise to a vial containing the 1 mlof chloroform lipid mixture. The vial was tightly sealedand vortex mixed for 6 minutes. Floating in a pool ofchloroform and excess lipids, spherules of water drop-lets coated with a lipid monolayer were produced.Next, 1 ml of this mixture was added rapidly to each ofthe two vials containing 2.5 ml of 240 /nmol/1 of glu-cose. The new suspension was vortexed for 4 seconds.The resulting liposomes were then dried in a flask con-taining 250 /xmol/1 of sucrose by passing nitrogen overthem at 8 1/min until no detectable chloroform re-mained. The nitrogen was then passed for an addi-tional 2 minutes. The mixture was centrifuged at 400X G for 10 minutes, producing a liposomal pellet. Toremove unencapsulated drug, the supernatant waswithdrawn and the liposomes were resuspended in0.9% sodium chloride solution and recentrifuged.This procedure was repeated five times to remove anyunencapsulated drug. Drug capture efficacy was de-termined by measuring the percentage of drugamount within liposomes versus that within liposomesand supernatant.

The drug release studies for ganciclovir and 2'-nor-cyclic GMP were performed in triplicate at 37°Cwith the liposomes suspended in phosphate bufferedsaline within a 30-ml syringe mounted on a revolvingrotor. At designated time intervals, 3 ml aliquots weredrawn off and centrifuged. The supernatant was then

TABLE l. Intravitreal Injections of 2'-nor-cyclic GMPDrugConcentration(iig/0.1 ml)

Control102050100

Day 1n/Hist/ERG

1/0/ND2/0/ND2/0/ND2/0/ND

Day 3n/Hist/ERG

1/0/ND2/0/ND1/0/ND2/0/ND2/1/ND

Day 7n/Hist/ERG

5/0/ND2/0/ND1/1/ND5/2/ND4/3/ND

Day 30n/Hist/ERG

2/0/03/0/0

3/3/32/3/3

Day 60n/Hist/ERG

1/0/02/0/02/0/02/3/31/3/3

n = number of eyes tested.Hist, histology. 0, no toxicity. 1, mild toxicity consisting of retinal pigment epithelium hyperplasia. 2, moderate toxicity consisting of vac-uolization of the retinal pigment epithelium and loss of height of the photoreceptor outer segment. 3, severe toxicily consisting ofdistortion of the entire outer retina with loss of retinal pigment epithelium and photoreceptor layer and reduction in the outer nuclear layer.ERG, electroretinogram, dark adapted. 0, normal waveform, amplitude, and latency. 3, severe loss of amplitude and abnormal waveforms.ND, not done. ERGs were not performed within 2 wk of injection because of nonspecific changes seen in our laboratory after intraocularinjections of any substance.1'* No data.


120

FIGURE l. Intravitreal injection of 10 /t/g 2'-nor-cyclic GMPin rabbit eye 2 months earlier. (A) Electroretinogram beforeand after (arrow) injection shows normal waveform, ampli-tude, and latency. (B) Light micrograph of methacrylate-em-bedded retina, the cytoarchitecture of the entire retina, ap-pears normal. G = ganglion cell layer, P = photoreceptorlayer, arrows = RPE. (Counterstained with toluidine blueX600). (C) Electron micrograph with high-power view of thephotoreceptor layer shows normal stacking of disks withminor artifactual splaying. The splaying of disk segmentsand separation of rods is identical to that seen in controlnoninjecled eyes. (Counterstained with lead citrate and ura-nyl acetate, X25,000). (D) Electron micrograph view of theouter nuclear layer (O) shows normal nuclear chromatinpattern and outer limiting membrane (arrows). (Counter-stained with lead citrate and uranyl acetate, X4,000). (E)Electron micrograph view of the inner ganglion layer showsinner limiting membrane (arrows). (Counterstained withlead citrate and uranyl acetate, X7,500).


Retinal Toxicity of the Anti-CMV Drug 2'-nor-cyclic GMP 2907

withdrawn. Next, the pelleted liposomes weredisrupted by freezing and resuspending in 1 ml dis-tilled water. Drug concentration was then determinedby high-pressure liquid chromatography for ganciclo-vir and high-pressure liquid chromatography and/or ultraviolet absorption at 254 nm for ^-nor-cyclic GMP.

Spectrophotometric analyses of 2'-nor-cyclic GMPwithin liposome fractions were performed by dilutingthe samples with chloroform-methanol (2:3 vol/vol)and measuring ultraviolet absorption at 254 nm. Theconcentration of 2'-nor-cyclic GMP was calculatedfrom the A-254 value, the molar absorbance of thecompound at 254 nm (13.7 X 103), and the molecularweight of the compound was 484.25. High-pressureliquid chromatographic analysis of either gancicloviror 2'-nor-cyclic GMP was performed by diluting an ali-quot of the chloroform methanol diluted sample andperforming chromatography on a carbon-18 columnfor ganciclovir or a Varian AX-10 analytical anion ex-change column for 2'-nor-cyclic GMP using isocraticelution with 80 nM KPO4 (pH 3.4) at a flow rate of 2ml/min for each drug. The amount of drug was deter-mined from the respective area under the peak thatwas read against a standard curve constructed by chro-matographing known amounts of each compoundunder the same conditions. Ganciclovir encapsulationwas carried out in the same fashion as 2'-nor-cyclicGMP with these modifications: 90 mM ganciclovir wasdissolved in sterile water and titrated with HCL to afinal pH of 1.2 before admixture with the lipid chloro-form preparation. In the drying phase, the sucrosesolution contained 40 mM of lysine and 210 mM ofsucrose.

RESULTS

All experiments were repeated two to four times (Ta-ble 1). The vitreous remained clear throughout thepostinjection period in all 25 rabbits. The 11 eyes thatreceived 10 fig of drug had normal ERG findings (am-plitude and latency) before and after the intraocularinjection at 30- and 60-day time points (Fig. 1). Lightmicroscopy and electron microscopy displayed normalretinal morphology at all time points (Figs. 2, 3, 4, 5).The retinas were observed to be attached and of nor-mal appearance in 10- and 20- .̂g doses. At the 20-figdose, the electroretinogram was normal at all timepoints. Mild changes consisting of vacuolization at thelevel of the retinal pigment epithelium were seen firstat 1 week by light microscopy; the histology had beennormal before this. At the 2-month time point, thesechanges resolved. The electroretinogram showed se-vere loss of amplitude after the first week after injec-tion at both the 50- and 100-jig doses. The retina lost

FIGURE 2. Light micrograph of methacrylate-embedded ret-ina of rabbit injected with 50 /ug of 2'-nor-cyclic GMP 1 weekearlier. The cytoarchilecture of the outer retina is markedlydistorted. The photoreceptor layer is markedly shortenedabove a hypertrophic RPE (between arrows). In one area(asterisk), the entire outer segment is lost and the outer nu-clear layer opposes Bruch's membrane. The ganglion andinner nuclear layers are unremarkable. (Counterstainedwith toluidine blue, X600).

transparency at 30 days. The 17 eyes that received 50and 100 jig showed toxic changes at 1 week, 1 month,and 2 months. Toxic changes observed at 50-jLtg dosesvaried from vacuolization of the retinal pigment epi-thelium and loss of height of the photoreceptor outersegments to severe loss of the entire outer retina.These were not seen 1 and 3 days after injection butbecame prominent at 1 week, and severe toxicity wasseen at the 1- and 2-month time points. At higherdoses (100 /xg), the toxic effects became evident at thelevel of the retinal pigment epithelium earlier (3 days)with destruction of the outer half to outer 75% of theretina seen, beginning at the 1-week time point.

Based on our studies, the intraocular therapeuticindex [maximum nontoxic dose (j/g/cc)/dose (/ig/cc)required to inhibit 50% HCMV colony formation] is10/.5 = 20.



FIGURE 3. Light micrograph of melhacrylate-ernbedded ret-ina of rabbit injected with 50 Mg of 2'-nor-cyclic GMP 1month earlier. The cytoarchitecture of the outer retina ismarkedly distorted. The photoreceptor layer and RPE areabsent. The outer nuclear layer is thinned and abuts Bruch'smembrane. The ganglion cell and outer nuclear layersare unremarkable. (Counterstained with toluidine blue,X1200).

Drug capture efficacy was 89% for 2'-nor-cyclicGMP within liposomes. The drug release half-life invitro was 1,000 hours for 2'-nor-cyclic GMP. Ganciclo-vir drug capture efficacy was 42%, and it had a drugrelease half-life of 13 hours in vitro.

DISCUSSION

CMV infections are a major cause of morbidity andmortality in the immunocompromised host. They maypresent as various pathologic entities, but retinitis isthe most common and requires indefinite treatment inthe patient with AIDS. DHPG (ganciclovir) is com-monly used in the treatment of CMV infections in theimmunocompromised patient but has many side ef-fects, particularly bone marrow toxicity. Anothercompound, phosphonoformate (foscarnet) has alsobeen used in the therapy of HCMV retinitis with good

success; however, the compound has disadvantagesthat include renal toxicity, anemia, electrolyte imbal-ance, tremor, and nausea. Recognizing that CMV reti-nitis may be diagnosed in the absence of systemic dis-ease and recognizing the disadvantages of systemic tox-icity and daily intravenous therapy with ganciclovir orfoscarnet, alleviation of these concerns might resultfrom use of local intravitreal preparations in the repo-sitory form which could be administered at infrequentintervals.

Although 2'-nor-cyclic GMP is a potent and selec-tive inhibitor of CMV replication in vitro, the com-pound's mode of antiviral action is unknown but mayinvolve inhibition of viral DNA polymerase. The ID50concentrations of ganciclovir and 2'-nor-cyclic GMP toinhibit HCMV infection in tissue culture are 0.43 7and 2 fimol/l, respectively.8'1 V|2J>22 Therefore, 2'-nor-

FIGURE 4. Light micrograph of methacrylate-embedded ret-ina of rabbit injected with 100 ng of 2'-nor-cyclic GMP 1month earlier. The cytoarchitecture of the outer retina ismarkedly distorted. The RPE and photoreceptor layer areabsent. The outer nuclear layer has been reduced to a singlecuboidal layer that abuts Bruch's membrane. Focally, theinner nuclear layer has collapsed into the outer nuclear layer(asterisk). The ganglion layer is unremarkable. (Counter-stained with toluidine blue, XI200).


Retinal Toxicity of the Anti-CMV Drug 2'-nor-cyclic GMP

150

0.09 0.18

Seconds0.27 0.36

FIGURE 5. Electroretinogram of rabbit before and after(arrow) 50-jug intravitreal injection of 2'-nor-cyclic GMPshows severe loss of amplitude and abnormal waveforms.

cyclic GMP and ganciclovir are comparably potentanti-CMV agents. Our results indicate that a 10-jLtgdose of 2'-nor-cyclic GMP, which is greater than 20times the concentration required to inhibit CMVplaque formation by 50%, is not toxic to the rabbitretina by light microscopy, electron microscopy, andelectrophysiologic testing for up to 8 weeks. Intra-vitreal injections of 2'-nor-cyclic GMP greater than 20^g/0.1 ml resulted in severe toxic effects on the outerretina, especially the photoreceptor layer. The ERGresults paralleled the morphologic findings at toxicand nontoxic doses at 1 month and 2 months. ERGsnot performed before this, because nonspecific ERGchanges were seen after intraocular injection of anysubstance.

We have shown that 2'-nor-cyclic GMP has a pro-longed release time in a multivesicular liposome sys-tem that can be used to increase the intraocular timeof residence of this compound. This will likely allowfor a more favorable toxicologic profile. Studies byour group have indicated that the multivesicular lipo-some system used in this study is not toxic to the rabbitretina. We have found that polar derivatives that arewater soluble and compounds of high molecularweight are well retained within a multivesicular lipo-some system. At neutral pH, ganciclovir is poorlywater soluble, and the encapsulation efficiency in theaqueous phase of liposomes is low. The safe intra-vitreal level of ganciclovir in rabbit is between 50 and200 jiig per injection.2324 Although ganciclovir is muchmore water soluble at pH II,22 the lipids used in lipo-somes are hydrolyzed at this pH, and injection ofhighly alkaline substances into the eye may be asso-ciated with toxic problems. Further studies of polardrugs such as 2'-nor-cyclic GMP encapsulated withinthe aqueous phase of liposomes are warranted because

2909

such compounds, suitably encapsulated in liposome,may prove to be a useful therapeutic modality for thelocal intravitreal therapy of CMV and other forms ofviral retinitis.

Key Words

antiviral drugs, CMV retinitis, AIDS, 2'-nor-cyclic GMP,retina

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19. Dolnak DR, Munguia D, Wiley CA, et al. Lack of reti-nal toxicity of the anti-CMV drug (S-l-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC). InvestOphthalmol Vis Sci. 1992;33:1557-1563.

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