Sodium hyaluronate as a carrier for intravitreal gentamicin an experimental study

69 (1991)45-49

ACTA 0 P H T H A L M 0 L O G ICA

Sodium hyaluronate as a carrier for intravitreal gentamicin an experimental study Carlos A. Moreira Jr’, Deborah K. Armstrong’, Roger W. Jelliffe’, Ana T. Moreira’, Coreen C. Woodford3, Peter E. Liggett’ and Melvin D. Trousdale’ Department of Ophthalmology, Doheny Eye Institute’,and Department of Medicine’, University of Southern California School of Medicine and University of Southern California School of Pharmacy3,Los Angeles, CA, USA

Abstract. Fifty mcg of gentamicin was combined with saline or with 0.8%sodium hyaluronate and injected into the vitreous cavity of rabbit eyes with moderate to severe Staphylococcus aureus endophthalmitis. Endophthalmitis was controlled in 9 of 10 eyes. There was no evidence of toxicity with either treatment regimen. Although the clearance study demonstrated statistical differences at all time points studied, the half-lives of both treatment regimens were similar (3.3 h for aqueous gentamicin and 3.6 h for sodium hyaluronate/gentamicin). These results suggest that the vitreous played a role in keeping the aqueous gentamicin in the eye for a longer time, as similar half-lives were shown with both types of treatment. Thus, if a vitrectomy has to be done for the treatment of endophthalmitis, as much as possible of the vitreous should be left in situ to maintain the drug for longer periods in the eye. Also, if it is necessary to remove all vitreous during vitrectomy, it may be more effective to administer the drug with sodium hyaluronate so as to prolong its action inside the eye. Key words: bacterial endophthalmitis - gentamicin - drug carrier - prophylactic antibiotic therapy - intraocular therapy.

Endophthalmitis remains one of the most disastrous complications of ophthalmic surgery and of accidental ocular trauma. The estimated incidence of such a complication following cataract extraction vanes from 0.02 to 0.96% (Allen 1978a;JaEe

1981), and the incidence after penetrating eye trauma is 8%(Brinton et al. 1984).The most commonly implicated bacterial pathogens are Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeurginosa (Allen 1978b; Brinton et al. 1984; Forster 1978). Despite the many advances in management of endophthalmitis, visual outcome remains poor (Bohigian & Olk 1986). Many studies report final visual results of 20/400 or better in 42 to 57%of the cases (Brinton et al. 1984; Forster et al. 1980; Olson et al. 1983; Rowsey et al. 1982; Vastine et al. 1979). Allen (1978b) reviewed 39 cases of endophthalmitis following cataract extraction and observed 67%with no light perception after treatment. The reasons for such poor results are not known, but may be due to virulence of the microorganism and consequent severe inflammatory response (Bohigian & Olk 1986), or to the fact that systemic, topical and subconjunctivaladministration of antibiotics may not provide adequate concentrations in the vitreous (Axelrod et al. 1985; Barza et al. 1983a;Peyman 1977;Rubinstein et al. 1983;Yannis et al. 1985).While the use of intravitreal antibiotics represents an inprovement in treatment (Baum et al. 1982; Diamond 1981; Kane et al. 1981; Peyman et al. 1977; Zachary & Forster 1976), the possibility of retinal toxicity from such drugs has been re45

ported at therapeutic concentrations (Conway 8c Campochiaro 1986; D'Amico et al. 1985;Talamo et al. 1985). Furthermore, the levels of antibiotics in infected eyes are lower than in normal eyes (Peyman et al. 1974). Kane et al. (1981) studied the clearance of antibiotics and noted that drug halflife is significantly shorter in inflamed eyes than in normal eyes, and suggested that more than one intravitreal injection of antibiotics is necessary to adequately treat endophthalmitis. The goals of the present study were to determine the effectivenessof intravitreal therapy using a combination of sodium hyaluronate and gentamicin in the treatment of bacterial endophthalmitis; to monitor for retinal toxicity; and to determine the effect of sodium hyaluronate on the clearance of gentamicin in an infected, aphakic, and partially vitrectomized rabbit eye.

Materialsand Methods Effectiveness New Zealand white rabbits (2.5 to 3 kg body weight) were inoculated bilaterally into the vitreous with 10' colony forming units of S. aureus (Moreira et al., accompanying paper). Twenty-four h later all animals were examined by a second observer who classified the severity of endophthalmitis using four grades: I) anterior chamber clear and mild haziness of the vitreous; 11) cells and flare in the anterior chamber and more haziness of the vitreous; 111) fibrin in the anterior chamber, no fundus view possible, but red reflex present; and IV) opaque cornea and absence of the fundus red reflex. Animals exhibiting only grades I1 and 111 were included in the complete study. Ten selected animals underwent pars plana lensectomy and a vitrectomy that did not exceed 30%of the total vitreous volume. In order to perform a 30% vitrectomy, the pars plana vitrectomy was done prior to the lensectomy and 0.4 ml of the vitreous was aspirated in a syringe. After placing all sutures, 0.3 ml of 50 mcg of aqueous gentamicin, with or without 0.8% sodium hyaluronate (molecular weight greater than 4 X lo"), was injected into the anterior portion of the vitreous cavity according to previous randomization. Preparation of gentamicin was done according to Moreira et al. (accompanying paper). The animals were examined daily for signs of ocular infection that would indicate wor-

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sening of the clinical status. Animals exhibiting these signs were sacrificed, and vitreous aspirates collected for culture identification of pathogens. Animals which did not exhibit worsening of the clinical status were sacrificed on the 10th postoperative day and vitreous aspirate collected for culture isolation and identification. Toxicity testing A toxic respone to sodium hyaluronate or gentamicin, or to a combination of the two, was monitored by indirect ophthalmoscopy, electroretinography (ERG) and histopathology. Five New Zealand white rabbits underwent the same surgery as described above, receiving intravitreally 50 mcg of gentamicin with sodium hyaluronate, but without previous S. aureus inoculation. The fellow nonmanipulated eye of each animal served as a negative control. Indirect ophthalmoscopy was performed every 3 days for up to 2 weeks after surgery. Settings for the ERG were 50 msechweep, with intensities of 1,4 and 16 (Mandelbaum et al. 1980).Animals were dark adapted for 15 min prior to the test. Following the ERG, the animals were sacrificed and the eyes enucleated for histopathologic studies. Clearance determination

Only animals with endophthalmitis of grades I1 and ILI severity were used for the clearance studies. They underwent the same type of surgery and treatment as previously described. The intraocular concentration and clearance of gentamicin, with and without sodium hyaluronate, were determined at 0, 2, 4, 8 and 12 h after injection of the antibiotic. At each time point, 4 animals were sacrificed; the eyes were enucleated, immediately placed in liquid nitrogen and stored at - 85°C. Eyes were removed from - 85°C storage and maintained for 1 h at -20°C prior to dissection. The fibrotic cover of the eye was removed and the frozen intraocular content was thawed, sonicated for 1 min, and then centrifuged for 10 min at 3500 rpm. Each supernatant was collected for quantification of gentamicin using a fluorescence polarization immunoassay (TDx System, Abbott Laboratories, Diagnostic Division, Irving, TX). The control and standard curve specimens included rabbit vitreous and basic salt solution mixed with known concentrations of gentamicin (intrathecal gentamicin, 4 mg/2 ml, Schering Phar-

maceutical Corporation, Manati, Puerto Rico). The standard curve dilutions were 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0 mcg/ml. The control gentamicin concentrations were 30, 10, 6 and 1.5 mcg/ml. They matched the fluorescence polarity of the control serum specimens at concentrations of 10,6 and 1.5 mcg/ml, which were provided by the manufacturer. The rabbit supernatants were assayed in duplicate for gentamicin concentrations. A t-test on population means for small sample sizes was used for statistical analysis of the continuous outcomes (DAmico et al. 1985).

Results Effectiveness

Only 1 of 10 eyes that received the intravitreal combination of sodium hyaluronate and gentamicin developed culture positive endophthalmitis after treatment. In the group that received gentamicin alone, 2 of 10 eyes had culture positive endophthalmitis after treatment. There was no increase in the intraocular pressure in either group. All other treated eyes did not present with endophthalmitis and were culture negative. There was no statistical difference between the treated groups ( P > 0.05).

Table 1.

Gentamicin concentrations with and without sodium hyaluronate in aphakic and vitrectomized rabbit eyes.

I Gentamicin concentrations (mcg/ml)

(h) Time"

0 2 4 8 12

Gentamicin (SD)b

11.95 4.38 5.32 3.38 1.87

(6.37) (5.04) (6.14) (1.57) (0.54)

Gentamicin/ SH (sn) 27.36 18.34 12.32 6.32 3.14

(4.53) (0.77) (3.59) (3.57) (1.06)

< 0.001 < 0.002