Pharmacokinetics, Tissue Localization, Toxicity, and Treatment Efficacy in the First Small Animal (Rabbit) Model of Intra-Arterial Chemotherapy for Retinoblastoma

Abstract

PurposeCurrent intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy.MethodsFollowing selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity.ResultsOA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 μM and area under the curve (AUC0→∞) was 5.26 μM·h. Treated eye vitreous Cmax was 2.24 μM and AUC0→∞ was 4.19 μM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 μM and AUC0→∞ was 2.07 μM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan.ConclusionsThis first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

Conservative management of retinoblastoma: Challenging orthodoxy without compromising the state of metastatic grace. “Alive, with good vision and no comorbidity”

“…Intra-arterial chemotherapy with melphalan or carboplatin has been shown in vivo to trigger vascular toxicity through endothelial cell inflammation and leukostasis, in a study without sham injection of saline to test the effect of the ophthalmic catheterization per se (Steinle et al, 2012). This was later tested in another animal model, demonstrating no vascular damage following saline injection (Daniels et al, 2018). The role of the micro-catheter positioning at the ostium versus occlusive engagement into the ophthalmic artery, as well as the continuous versus pulsatile injection mode, have also been debated.…”

“…Finally, various tumor and non-tumor bearing animal models have been used to study the pharmacokinetic of different drugs depending on the intraocular delivery model. Large animals such as primates, pigs and rabbits are preferentially chosen because of their appropriate size, their similar proportion between ocular compartments compared to human eyes and the possibility they allow for repeated or continuous sampling of the ocular fluids and the cannulation of the ophthalmic arteries (Carcaboso et al, 2007(Carcaboso et al, , 2010Daniels et al, 2018;Ditta et al, 2012;Schaiquevich et al, 2012c;Vézina, 2013). Macaques and rabbits have been successfully used to characterize the toxicity of intra-ophthalmic artery and intravitreal melphalan in the retina (Francis et al, Fundus showing nasal incomplete choroidal atrophy and preserved temporal choroid.…”

“…); n Dose: 5 μg(Buitrago et al, 2010); o Dose:7 mg (Schaiquevich et al, 2012b); p Dose: 15 μg (Buitrago et al, 2016); q Dose: 0.4 mg/kg(Daniels et al, 2018).…”

Development of sheet‐enhanced technique (Set) method for matrix‐assisted laser desorption/ionization imaging mass spectrometry

“…Therefore, MALDI‐IMS has been developed to reveal the distribution of biomolecules, such as phospholipids, proteins, glycolipids, and oligonucleotides . MALDI‐IMS is also used for pharmacokinetic analyses, to visualize a parent drug and its metabolites simultaneously …”

“…12,13 MALDI-IMS is also used for pharmacokinetic analyses, to visualize a parent drug and its metabolites simultaneously. [14][15][16] The key to successful experiments in MALDI-IMS is to apply the matrix uniformly to the sample since the ratio of matrix to molecule greatly affects ionization. Matrix deposition methods include nebulized spray coating, 17 electrospray deposition, 18 automated acoustic deposition, 19 matrix sublimation, 20 piezoelectric-based matrix inkjet printers, 21 spray-droplet deposition, 22 and vapor deposition coupled with matrix recrystallization.…”