| Background and Aims Fungal keratitis is the first common infectious corneal diseases and has become the leading cause of blindness resulting from corneal diseases in China. Current treatment protocols for mycotic infections are far from optimal, due to a combination of the growth characteristics of fungi, late diagnosis, scarcity of effective antifungal agents and poor tissue penetration of the currently available therapeutic agents.Amphotericin B (AMB), a macrocyclic polyene, is a first-line treatment of keratitis caused by Candida species in many countries and for fungal keratitis in regions where natamycin is not available. It is also active against Aspergillus species and Fusarium species. Intracameral or intracorneal AMB can achieve significant therapeutic efficacy in the treatment of recalcitrant fungal keratitis not responding to conventional antifungal therapy. The intracameral or intracorneal injection is simple and less invasive. However, to date, few report could be found in the literature discussing the corneal toxicity of intracorneal injection of AMB or corneal distribution of AMB following intracameral or intracorneal injection.The aim of our study was to investigate the toxicity to cornea of intrastromal injection with AMB in rabbits, explore a safe range of antibiotic concentration, and investigate the concentrations of AMB in the cornea and aqueous humor following 3 different administrations, 0.25% AMB topical application(debribed cornea), intrastromal and intracameral injection of AMB ( 10μg per 0.1ml ) in a rabbit noninfected model. We sought to provide the laboratory rationale for the administration selection of the keratomycosis therapy and administration time interval.Part 1 Intrastromal injection of amphotericin B in rabbits eyes:corneal toxcity?Objective To investigate the toxicity to cornea of intrastromal injection with AMB in rabbits, and explore a safe range of antibiotic concentration. Methods1. Totally 20 healthy rabbits (40 eyes) were randomly divided into 5 groups with 8 eyes in each group.2. The eyes of Group A received intrastromal injection of 0.1ml balanced salt solution, and those of Group B,C,D and E received 0.1ml injection containing AMB 5,10,15,and 20μg respectively.3. Every rabbit received 5 intrastromal injections, once per 4 d. In 1d, 3d after each injection, corneal transparency, epithelial defect and corneal neovascularization were observed with slit lamp biomicroscopy, and corneal thickness was measured with ultrasonic pachymetry. In 30 d after the last injection, all animals were sacrificed. Their corneas were removed. Pathological observations were carried out for corneal morphology, survival rate of the corneal endothelial cells and ultrastructure of corneal endothelium.Results1. In the rabbits of intrastromal injection of AMB at concentrations of 5μg per 0.1ml and 10μg per 0.1ml, they had transparent and intact cornea, little corneal neovascularization, normal endothelial cells, and had no any significant difference with those of Group A(P>0.05).2. However, in the rabbits of intrastromal injection of AMB at concentrations of 15μg per 0.1ml and 20μg per 0.1ml, the rabbits showed edema cornea, exfoliation of corneal epithelium and severe corneal neovascularization, with significant difference compared with Group A(P<0.05).Conclusions1. Intrastromal injection is simple, less invasive, can directly deliver drug into the deep cornea.2. Intrastromal injection of AMB at the concentration of 5μg per 0.1ml or 10μg per 0.1ml appears to show no toxicity to rabbit corneal keratocytes or endothelial cells.3. Intrastromal injection of AMB at the concentration of 15μg per 0.1ml or 20μg per 0.1ml appears to be deleterious to rabbit cornea, if the injection frequency is high. Part 2 Corneal and aqueous humor concentrations following 3 different administrations of amphotericin B in a rabbit modelObjective To investigate the AMB concentrations in the cornea and aqueous humor of rabbits following 3 different administrations of topical 0.25% AMB eye drop, intrastromal and intracameral injection of AMB ( 10μg per 0.1ml ).Methods1. 45 healthy rabbits were randomly divided into three groups with 15 rabbits in each group.2. Intrastromal and intracameral injection of 0.1ml AMB (10μg per 0.1ml) were administered in group A and group B separately, topical 0.25% AMB (50μl per a drop, once per 5 minutes, 6 doses together) was administered in group C (corneal epithelium debrided).3. The cornea and aqueous humor were obtained at 30min, 6h, 1d, 3d and 7d after applications.All samples were analyzed by high-performance liquid chromatography (HPLC).4. The HPLC system comprised a Waters model 510 pump, Hypersil C18 column (200.0mm×4.6mm,5.0μm). The UV detector was set to 380.0 nm. Acetonitrile-0.01mol/l NaH2PO4(35:65,V/V) containing 0.05ml triethylamine was used as mobile phase and the flow rate was 1.0ml/min. External standard was used in this assay.Results1. Calibration curves were linear over the range of 0.10～100.00μg/ml.The concentration at 0.10μg/ml was the lowest quantified limit. The recovery of AMB from aqueous humor samples ranged from 89.1%～95.7%, and ranged from 81.4%～83.6% in the cornea samples.2. After single application, effective high drug levels were achieved in cornea in Group A, maintained 7d, exceeded the minimum inhibitory concentration at which 90% of isolates are inhibited(MIC90) for a wide spectrum of fungi and mold, including Aspergillus, Fusarium, and Candida species. There were significant differences compared with group B and group C( P<0.05). 3. Effective high drug levels were achieved in the aqueous humor in Group B, exceeded MIC90 for a wide spectrum of fungi and mold at 30 min, but decreased abruptly within 1 d. There were significant differences compared with group A and group C(P<0.05).4. The concentrations in corneas and aqueous increased after frequent topical administration of AMB (cornea debrided).Conclusions1. High concentrations of AMB were achieved in the rabbit cornea after intrastromal injection, maintained 7 d. So repeat intrastromal injection should be scheduled in 7d.2. Intracameral injection could enhance the concentrations in the aqueous humor, but it decreased abruptly within 1d.So repeat injection next day may be necessary to acute invasive keratomycosis.3. During early stage of fungal keratitis, epithelium debrided, 0.25% AMB topical application may be a good selection.4. Intrastromal or intracameral injection of AMB may be a therapeutic adjunctive for the management of deep recalcitrant fungal keratitis. |
