(±)Modafinil ((±)MOD) and its R-enantiomer (R-modafinil; R-MOD) have been investigated for their potential as treatments for psychostimulant addiction. We recently reported a series of (±)MOD analogs, of which JJC8-016 (N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)-3-phenylpropan-1-amine) was selected for further development. JJC8-016 and R-MOD were evaluated for binding across ~70 receptors, transporters, and enzymes. Although at a concentration of 10 μM, there were many hits for JJC8-016, binding affinities in the range of its DAT affinity were only observed at the serotonin transporter (SERT), dopamine D-like, and sigma receptors. R-MOD was more selective, but had much lower affinity at the DAT (K=3 μM) than JJC8-016 (K=116 nM). In rats, systemic administration of R-MOD alone (10-30 mg/kg i.p.) dose-dependently increased locomotor activity and electrical brain-stimulation reward, whereas JJC8-016 (10-30 mg/kg i.p.) did not produce these effects. Strikingly, pretreatment with JJC8-016 dose-dependently inhibited cocaine-enhanced locomotion, cocaine self-administration, and cocaine-induced reinstatement of drug-seeking behavior, whereas R-MOD inhibited cocaine-induced reinstatement only at the high dose of 100 mg/kg. Notably, JJC8-016 alone neither altered extracellular dopamine in the nucleus accumbens nor maintained self-administration. It also failed to induce reinstatement of drug-seeking behavior. These findings suggest that JJC8-016 is a unique DAT inhibitor that has no cocaine-like abuse potential by itself. Moreover, pretreatment with JJC8-016 significantly inhibits cocaine-taking and cocaine-seeking behavior likely by interfering with cocaine binding to DAT. In addition, off-target actions may also contribute to its potential therapeutic utility in the treatment of cocaine abuse.

Short-term improvements in retinal anatomy are known to occur in preclinical models of photoreceptor transplantation. However, correlative changes over the long term are poorly understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using noninvasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial evaluation of photoreceptor transplantation over the long term. Methods: Photoreceptor cell suspensions or sheets from rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-Prkdc scid /J or C3H/HeJ-Pde6b rd1 mice. Multimodal cSLO imaging was performed serially for up to three months after transplantation. Imaging biomarkers were scored, and a grade was defined for each eye by integrating the scores. Image grades were correlated with immunohistochemistry (IHC) data. Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, hemorrhage, retinal atrophy, and periretinal proliferation. Migration of transplanted material was observed. Changes in biomarker scores and grades were detected in 14/16 and 7/16 eyes, respectively. A high correlation was found between image grades and IHC parameters. Conclusions: Serial evaluation of multiple imaging biomarkers, when integrated into a per-eye grading scheme, enabled comprehensive tracking of longitudinal changes in photoreceptor cell grafts over time. The application of systematic multimodal in vivo imaging could be useful in increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other animal models. Translational Relevance: By allowing longitudinal evaluation of the same animal over time, and providing quantifiable biomarkers, non-invasive multimodal imaging improves the efficiency of retinal transplantation studies in animal models. Such assays will facilitate the development of cell therapy for retinal diseases.

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32Purpose: Short-term improvements in retinal anatomy are known to occur in preclinical models 33 of photoreceptor transplantation. However, correlative changes over the long term are poorly 34 understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using non-35 invasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial 36 evaluation of photoreceptor transplantation over the long term. 37Methods: Yellow-green fluorescent microspheres were transplanted into the vitreous cavity 38 and/or subretinal space of C57/BL6J mice. Photoreceptor cell suspensions or sheets from 39 rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-40 Prkdc scid /J or C3H/HeJ-Pde6b rd1 mice. Multimodal cSLO imaging was performed serially for up 41 to three months after transplantation. Imaging biomarkers were scored, and a grade was defined 42 for each eye by integrating the scores. Image grades were correlated with immunohistochemistry 43 (IHC) data. 44 Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers 45 including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, 46 hemorrhage, retinal atrophy, and peri-retinal proliferation. Migration of transplanted material 47 increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other 53 animal models. 54 Key words: degenerative retinal diseases, age-related macular degeneration, stem cell therapy, 55 xenotransplantation, photoreceptor cell, retinal organoid, confocal scanning laser 56 ophthalmoscopy 57 Photoreceptor transplantation is being developed as a therapeutic modality to restore vision in 59 people affected by retinal degenerative diseases, including retinitis pigmentosa (RP) and age-60 related macular degeneration (AMD) 1-6 . Short term improvements in outer retinal anatomy after 61 photoreceptor cell transplantation have been observed, mainly by histological staining in 62 preclinical models of retinal degeneration 4,7 . However, histology is a relatively inefficient 63 method to track graft and recipient anatomy longitudinally over the long term. Histological 64 assays are labor-intensive, require large initial cohorts of recipients, and face the challenge of 65 recipient attrition over time. Non-invasive imaging could facilitate the longitudinal evaluation of 66 retinal anatomy in relatively smaller cohorts of recipient animals over time, without the need to 67 sacrifice animals at every assessment time point. 68 Recent advances in imaging techniques have enabled detailed imaging studies in mouse models 69 of retinal disease and regeneration 8-10 . Confocal scanning laser ophthalmoscopy (cSLO) can be 70 used to capture images in multiple imaging modes, including short-wavelength fluorescence 71 (SWF) excitation (488 nm) to detect photoreceptor cells labeled with green fluorescent protein 72 (GFP) in mouse recipients 1 . Multicolor reflectance (MR) imaging combines blue (488 nm)...

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