Pharmacokinetics of Melphalan After Intravitreal Injection in a Rabbit Model.

PURPOSE: Although widely used for vitreous seed control in retinoblastoma patients, currently there are no data on melphalan pharmacokinetics after intravitreal injections. Therefore, in this study, we characterized the ocular and systemic disposition of melphalan after intravitreal injection in the rabbit eye. METHODS: New Zealand rabbits received a single intravitreal injection of 15 µg of melphalan. Vitreous, aqueous, retina, and blood samples were collected at different times up to 12 h after the injection. Melphalan was quantitated in the biological samples using a validated high-performance liquid-chromatography technique and pharmacokinetic parameters were calculated by means of compartmental models. RESULTS: Model-predicted melphalan maximum vitreous, aqueous, and retina concentrations were 7.8 µg/mL, 0.024 µg/mL, and 9.8 µg/g tissue, respectively, attained immediately and at 0.8 and 0.25 h after intravitreal injection. Melphalan vitreous concentrations were higher than 0.3 µg/mL for 5 h after dosing. The elimination half-life from the vitreous, aqueous humor, and retina was 1.0, 0.2, and 1.2 h, respectively. Aqueous exposure [area under the curve (AUC)] was only 0.7% of that of the vitreous AUC. Melphalan concentrations in the retina were still detectable 12 h after dosing, while plasma exposure was under the limit of quantitation. CONCLUSION: Intravitreal administration of 15 µg melphalan leads to pharmacological vitreous levels with low aqueous exposure. Melphalan concentrations in the retina were measurable up to 12 h after dosing, but we report nondetectable systemic exposure in the rabbit. The results correlate with the clinical features of retinoblastoma patients that show control of vitreous seeds without systemic toxicity using intravitreal melphalan.

Pharmacokinetics, Safety, and Efficacy of Intravitreal Digoxin in Preclinical Models for Retinoblastoma.

PURPOSE: To assess in vitro cytotoxic activity and antiangiogenic effect, ocular and systemic disposition, and toxicity of digoxin in rabbits after intravitreal injection as a potential candidate for retinoblastoma treatment. METHODS: A panel of two retinoblastoma and three endothelial cell types were exposed to increasing concentrations of digoxin in a conventional (72-hour exposure) and metronomic (daily exposure) treatment scheme. Cytotoxicity was defined as the digoxin concentration that killed 50% of the cells (IC50) and was assessed with a vital dye in all cell types. Induction of apoptosis and cell-cycle status were evaluated by flow cytometry after both treatment schemes. Ocular and systemic disposition after intravitreal injection as well as toxicity was assessed in rabbits. Electroretinograms (ERGs) were recorded before and after digoxin doses and histopathological examinations were performed after enucleation. RESULTS: Digoxin was cytotoxic to retinoblastoma and endothelial cells under conventional and metronomic treatment. IC50 was comparable between both schedules and induced apoptosis in all cell lines. Calculated vitreous digoxin Cmax was 8.5 µg/mL and the levels remained above the IC50 for at least 24 hours after intravitreal injection. Plasma digoxin concentration was below 0.5 ng/ml. Retinal toxicity was evident after the third intravitreal dose with considerable changes in the ERG and histologic damage to the retina. CONCLUSIONS: Digoxin has antitumor activity for retinoblastoma while exerting antiangiogenic activity in vitro at similar concentrations. Metronomic treatment showed no advantage in terms of dose for cytotoxic effect. Four biweekly injections of digoxin led to local toxicity to the retina but no systemic toxicity in rabbits.

Ocular pharmacology of topotecan and its activity in retinoblastoma.

PURPOSE: To review the ocular pharmacology and antitumor activity of topotecan for the treatment of retinoblastoma by an evaluation of different routes of administration. METHODS: Systematic review of studies available at PubMed using the keywords retinoblastoma, topotecan, and camptothecins, including preclinical data such as cell lines and animal models, as well as clinical studies in patients with retinoblastoma. RESULTS: Forty-two available studies were reviewed. Evidence of antitumor activity against retinoblastoma as a single agent is based on data on cell lines and a limited number of affected patients with intraocular and extraocular disease when given in a protracted schedule. Evidence of additive or synergistic activity in combination with other agents such as carboplatin, melphalan, and vincristine was reported in preclinical and clinical models. In animal models, pharmacokinetic evaluation of topotecan administered by the periocular route shows that most of the drug reaches the vitreous through the systemic circulation. Topotecan administered by intravitreal injection shows high and sustained vitreal concentrations with limited systemic exposure and lack of retinal toxicity at a dose of up to 5 µg. Topotecan administered intraophthalmic artery shows higher passage to the vitreous compared with periocular administration in a swine model. CONCLUSION: Topotecan alone or in combination is active against retinoblastoma. It shows a favorable passage to the vitreous when given intravenously and intraarterially, and ocular toxicity is minimal by all routes of administration. However, its clinical role, optimal dose, and route of administration for the treatment of retinoblastoma are to be determined.

Local and systemic toxicity of intravitreal melphalan for vitreous seeding in retinoblastoma: a preclinical and clinical study.

PURPOSE: Intravitreal melphalan is emerging as an effective treatment for refractory vitreous seeds in retinoblastoma, but there is limited understanding regarding its toxicity. This study evaluates the retinal and systemic toxicity of intravitreal melphalan in retinoblastoma patients, with preclinical validation in a rabbit model. DESIGN: Clinical and preclinical, prospective, cohort study. PARTICIPANTS: In the clinical study, 16 patient eyes received 107 intravitreal injections of 30 µg melphalan given weekly, a median of 6.5 times (range, 5-8). In the animal study, 12 New Zealand/Dutch Belt pigmented rabbits were given 3 weekly injections of 15 µg of intravitreal melphalan or vehicle to the right eye. METHODS: Electroretinogram (ERG) responses were recorded in both humans and rabbits. For the clinical study, ERG responses were recorded at baseline, immediately before each injection, and at each follow-up visit; 82 of these studies were deemed evaluable. Median follow-up time was 5.2 months (range, 1-11). Complete blood counts (CBCs) were obtained on the day of injection at 46 patient visits. In the animal study, ERG responses were obtained along with fluorescein angiography, CBCs, and melphalan plasma concentration. After humane killing, the histopathology of the eyes was evaluated. MAIN OUTCOME MEASURES: For the clinical study, we measured peak-to-peak ERG amplitudes in response to 30-Hz photopic flicker stimulation with comparisons between ERG studies before and after intravitreal melphalan. For the animal study, we collected ERG parameters before and after intravitreal melphalan injections with histopathologic findings. RESULTS: By linear regression analysis, over the course of weekly intravitreal injections in retinoblastoma patients, for every additional injection, the ERG amplitude decreased by approximately 5.8 µV. The ERG remained stable once the treatment course was completed. In retinoblastoma patients, there were no grade 3 or 4 hematologic events. One week after the second injection in rabbits, the a- and b-wave amplitude declined significantly in the melphalan treated eyes compared with vehicle-treated eyes (P<0.05). Histopathology revealed severely atrophic retina. CONCLUSIONS: Weekly injections of 30 µg of melphalan can result in a decreased ERG response, which is indicative of retinal toxicity. These findings are confirmed at an equivalent dose in rabbit eyes by ERG measurements and by histopathologic evidence of severe retinal damage. Systemic toxicity with intravitreal melphalan at these doses in humans or rabbits was not detected.

Clinical pharmacokinetics of intra-arterial melphalan and topotecan combination in patients with retinoblastoma.

PURPOSE: To assess the antitumor activity, toxicity, and plasma pharmacokinetics of the combination of melphalan and topotecan for superselective ophthalmic artery infusion (SSOAI) treatment of children with retinoblastoma. DESIGN: Single-center, prospective, clinical pharmacokinetic study. PARTICIPANTS: Twenty-six patients (27 eyes) with intraocular retinoblastoma. METHODS: Patients with an indication for SSOAI received melphalan (3-6 mg) and topotecan (0.5-1 mg; doses calculated by age and weight). Plasma samples were obtained for pharmacokinetic studies, and a population approach via nonlinear mixed effects modeling was used. Safety and efficacy were assessed and compared with historical cohorts of patients treated with melphalan single-agent SSOAI. MAIN OUTCOME MEASURES: Melphalan and topotecan pharmacokinetic parameters and efficacy and safety parameters. RESULTS: Twenty-seven eyes from 26 consecutive patients received 66 cycles of SSOAI melphalan and topotecan in combination. All 5 eyes treated as primary therapy responded to the combination chemotherapy and were preserved. Sixteen of the 22 eyes with relapsed or resistant tumors responded, but 3 of them ultimately underwent enucleation at a median of 8 months (range, 7.9-9.1 months). The incidence of grade III and IV neutropenia was 10.6% and 1.5%, respectively, which was comparable with historical controls of single-agent SSOAI melphalan. No episode of fever neutropenia was observed, and no patient required transfusion of blood products. The large variability in melphalan pharmacokinetics was explained by body weight (P <0.05). Concomitant topotecan administration did not influence melphalan pharmacokinetic parameters. There was no effect of the sequence of melphalan and topotecan administration in plasma pharmacokinetics. CONCLUSIONS: A regimen combining melphalan and topotecan for SSOAI treatment of retinoblastoma is active and well tolerated. This combination chemotherapy previously showed synergistic pharmacologic activity, and we herein provide evidence of not increasing the hematologic toxicity compared with single-agent melphalan.

Ocular and systemic toxicity of intravitreal topotecan in rabbits for potential treatment of retinoblastoma.

Treatment of intraocular retinoblastoma with vitreous seeding is a challenge. Different routes of chemotherapy administration have been explored in order to attaining pharmacological concentrations into the posterior chamber. Intravitreal drug injection is a promissing route for maximum bioavailability to the vitreous but it requires a well defined dose for achieving tumor control while limited toxicity to the retina. Topotecan proved to be a promising agent for retinoblastoma treatment due to its pharmacological activity and limited toxicity. High and prolonged concentrations were achieved in the rabbit vitreous after 5 µg of intravitreal topotecan. However, whether a lower dose could achieve potentially therapeutic levels remained to be determined. Thus, we here study the pharmacokinetics of topotecan after 0.5 µg and the toxicity profile of intravitreal topotecan in the rabbit eye as a potential treatment of retinoblastoma. A cohort of rabbits was used to study topotecan disposition in the vitreous after a single dose of 0.5 µg of intravitreal topotecan. In addition, an independent cohort of non-tumor bearing rabbits was employed to evaluate the clinical and retinal toxicity after four weekly injections of two different doses of intravitreal topotecan (Group A, 5 µg/dose; Group B, 0.5 µg/dose) to the right eye of each animal. The same volume (0.1 ml) of normal saline was administered to the left eye as control. A third group of rabbits (Group C) served as double control (both eyes injected with normal saline). Animals were weekly evaluated for clinical and hematologic values and ocular evaluations were performed with an inverse ophthalmoscope to establish potential topotecan toxicity. Weekly controls included topotecan quantitation in plasma of all rabbits. Electroretinograms (ERGs) were recorded before and after topotecan doses. One week after the last injection, topotecan concentrations were measured in vitreous of all eyes and samples for retinal histology were obtained. Our results indicate that topotecan shows non linear pharmacokinetics after a single intravitreal dose in the range of 0.5-5 µg in the rabbit. Vitreous concentration of lactone topotecan was close to the concentration assumed to be therapeutically active after 5 h of 0.5 µg intravitreal administration. Eyes injected with four weekly doses of topotecan (0.5 or 5 µg/dose) showed no significant differences in their ERG wave amplitudes and implicit times in comparison with control (p > 0.05). Animals showed no weight, hair loss or significant changes in hematologic values during the study period. There were no significant histologic damage of the retinas exposed to topotecan treatments. After intravitreal administration no topotecan could be detected in plasma during the follow-up period nor in the vitreous of treated and control animals after 1 week of the last injection. The present data shows that four weekly intravitreal injection of 5 µg of topotecan is safe for the rabbit eye. Despite multiple injections of 0.5 µg of topotecan are also safe to the rabbit eye, lactone topotecan vitreous concentrations were potentially active only after 5 h of the administration. We postulate promising translation to clinics for retinoblastoma treatment.

Pharmacokinetic analysis of melphalan after superselective ophthalmic artery infusion in preclinical models and retinoblastoma patients.

PURPOSE: To characterize melphalan pharmacokinetics after superselective ophthalmic artery infusion (SSOAI) in animals and children with retinoblastoma. METHODS: Vitreous and plasma samples of five Landrace pigs were obtained over a 4-hour period after SSOAI of melphalan (7 mg). Melphalan cytotoxicity was evaluated in retinoblastoma cell lines with and without topotecan. Plasma samples were obtained from 17 retinoblastoma patients after SSOAI of 3 to 6 mg of melphalan to one (n=14) or two eyes (n=3). Correlation between plasma pharmacokinetics and age, dosage, and systemic toxicity was studied in patients. RESULTS: In animals, melphalan peak vitreous levels were greater than its IC50 and resulted in 3-fold vitreous-to-plasma exposure. In patients, a large variability in pharmacokinetic parameters was observed and it was explained mainly by body weight (P<0.05). A significantly higher systemic area under the curve was obtained in children receiving more than 0.48 mg/kg for bilateral tandem infusions (P<0.05). These children had 50% probability of grades 3-4 neutropenia. Plasma concentrations after 2 and 4 hours of SSOAI were significantly higher in these children (P<0.05). A synergistic cytotoxic effect of melphalan and topotecan was evident in cell lines. CONCLUSIONS: Potentially active levels of melphalan after SSOAI were achieved in the vitreous of animals. Low systemic exposure was found in animals and children. Doses greater than 0.48 mg/kg, given for bilateral tandem infusions, were associated with significantly higher plasma levels and increased risk of neutropenia. Synergistic in vitro cytotoxicity between melphalan and topotecan favors combination treatment.

Pharmacokinetic analysis of topotecan after superselective ophthalmic artery infusion and periocular administration in a porcine model.

PURPOSE: To characterize the vitreous and plasma pharmacokinetics of topotecan after ophthalmic artery infusion (OAI) subsequent to superselective artery catheterization and to compare it with periocular injection (POI). METHODS: The ophthalmic artery of 4 pigs was catheterized and 1 mg of topotecan infused over a period of 30 minutes. The contralateral eye was subsequently used for administering topotecan by POI. Serial vitreous specimens were obtained by microdialysis and plasma samples collected and assayed for total and lactone topotecan. RESULTS: Maximum total topotecan concentration in the vitreous (median, range) was significantly higher after OAI compared with POI (131.8 ng/mL [112.9-138.7] vs. 13.6 ng/mL [5.5-15.3], respectively; P < 0.005). Median vitreous exposure calculated as area under the curve for total topotecan attained after OAI was significantly higher than after POI (299.8 ng·hour/mL [247.6-347.2] and 48.9 ng·hour/mL [11.8-63.4], respectively; P < 0.05). The vitreous to plasma exposure ratio was 29 after OAI and 3.4 after POI. Systemic exposure for total topotecan was low after both modalities of administration, with a trend to be lower after OAI compared with POI (10.6 ng·hour/mL [6.8-13.4] vs. 18.7 ng·hour/mL [6.3-21.7]; P = 0.54). CONCLUSION: Superselective OAI resulted in significantly higher vitreous concentrations and exposure and a trend toward lower systemic exposure than POI.

Pharmacokinetic analysis of topotecan after intra-vitreal injection. Implications for retinoblastoma treatment.

Topotecan is a promising drug with activity against retinoblastoma, however, attaining therapeutic concentrations in the vitreous humor is still a challenge for the treatment of vitreous seeds in retinoblastoma. Our aim was to characterize topotecan pharmacokinetics in vitreous and aqueous humor, and to assess the systemic exposure after intra-vitreal injection in rabbits as an alternative route for maximizing local drug exposure. Anesthetized rabbits were administered intra-vitreal injections of 5 microg of topotecan. Vitreous, aqueous, and blood samples were collected at pre-defined time points. A validated high-performance liquid chromatography assay was used to quantitate topotecan (lactone and carboxylate) concentrations. Topotecan pharmacokinetic parameters were determined in vitreous, aqueous and plasma using a compartmental analysis. Topotecan lactone concentrations in the vitreous of the injected eye were about 8 ng/mL 48 h after drug administration. The median maximum vitreous, aqueous and plasma total topotecan concentrations (C(max)) were 5.3, 0.68 and 0.21 microg/mL, respectively. The C(max) vitreous/aqueous of treated eyes and the C(max) vitreous/plasma were approximately 8 and 254, respectively. Total topotecan exposure (AUC) in the vitreous of the injected eye was 50 times greater than the total systemic exposure. These findings suggest that intra-vitreal administration of only 5 microg of topotecan reaches significant local levels over an extended period of time while minimizing systemic exposure in the rabbit. Intra-vitreal topotecan administration offers a promising alternative route for enhanced drug exposure in the vitreous humor with potential application for treatment of vitreal seeds in retinoblastoma while avoiding systemic toxicities.