Enhanced Brain Delivery of Dimethyl Fumarate Employing Tocopherol

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Enhanced Brain Delivery of Dimethyl Fumarate employing Tocopherol Acetate-based Nanolipidic Carriers: Evidences from Pharmacokinetic, Biodistribution and Cellular Uptake Studies Pramod Kumar, Gajanand Sharma, Rajendra Kumar, Ruchi Malik, Bhupinder Singh, Om Prakash Katare, and Kaisar Raza ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00428 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016

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ACS Chemical Neuroscience

Enhanced Brain Delivery of Dimethyl Fumarate employing Tocopherol Acetate-based Nanolipidic Carriers: Evidences from Pharmacokinetic, Biodistribution and Cellular Uptake Studies Pramod Kumar1, Gajanand Sharma2, Rajendra Kumar3, Ruchi Malik1, Bhupinder Singh2, 3, O. P. Katare and Kaisar Raza1,* 1

Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandar Sindri, Distt. Ajmer, Rajasthan, India-305817. 2

Division of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India-160014 3

UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites, Panjab University, Chandigarh, India-160 014. .

*Address of correspondence Dr. Kaisar Raza Department of Pharmacy School of Chemical Sciences &Pharmacy Central University of Rajasthan Bandar Sindri, Distt. Ajmer, Rajasthan, India-305 817 E-mail: [email protected]; [email protected]

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Table for contents use only Manuscript title: Enhanced Brain Delivery of Dimethyl Fumarate employing Tocopherol Acetate-based Nanolipidic Carriers: Evidences from Pharmacokinetic, Biodistribution and Cellular Uptake Studies Authors: Pramod Kumar, Gajanand Sharma, Rajendra Kumar, Ruchi Malik, Bhupinder Singh, O. P. Katare and Kaisar Raza


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Abstract Dimethyl fumarate (DMF) is an approved drug for the management of relapsing multiple sclerosis. Despite efficacy, DMF is also reported to be a challenging drug owing to concerns like GIT flushing, multiple dosing, lower brain permeability, less patient compliance and economic hurdles. The present study aims to develop DMF-tocopherol acetate nanolipidic carrier (NLCs) to enhance brain permeability and improve the gastric tolerance. The developed DMF-tocopherol acetate NLCs offered an average size of 69.70 nm, PDI of 0.317 and a zeta potential of −9.71 mV. Higher drug entrapment (90.12%) and drug loading (20.13 %) assured controlled drug release behaviour both in gastric and intestinal pH. Cellular uptake studies on Caco-2 and SH-SY5Y monolayers confirmed better intestinal absorption and neuronal uptake of the developed system, which was further corroborated by the pharmacokinetic and biodistribution studies. The oral bioavailability was enhanced by 4.09 times and brain availability was substantially improved vis-à-vis plain drug. The findings are promising and offer preclinical evidence for better brain availability of DMF, which can be exploited in the better management of diseases like multiple sclerosis. Keywords: Caco-2, drug delivery, vitamin E, SLNs, NLCs, SH-SY5Y, confocal laser scanning microscopy



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1. Introduction Neurological disorders have been reported as the third cause of mortality worldwide (12%), following cardiovascular (31%) and cancer diseases (13%), as per WHO 1. MS is a chronic autoimmune neurological disorder, which is marked by demyelination of the neurons 2. Approximately 2.5 million global population comprising of age group of 20-40 years is reported to be affected by MS 3. Out of the four known types of MS, 85% of MS patients suffer from relapsing MS 4. Though the etiology of relapsing MS is still not fully understood, however, theories of oxidative stress and neuronal damage are being frequently forwarded as one of the reasons behind it 5. Relapsing MS is marked by disability in walking and coordination 6. Approx. 08 formulations have been marketed for the management of relapsing MS, including interferon beta, glatiramer acetate, teriflunomide, dimethyl fumarate (DMF), natalizumab, fingolimod, mitoxantrone and almtuzumab 2. Fat soluble vitamins are also reported for remyelination of neurons in MS 6,7. DMF, a fumaric acid derivative, has been extensively used for the management of psoriasis since 1990 by both oral and topical routes 8. Somewhere in 2013-14, DMF has been established and approved for the MS management, owing to the immune modulation and remyelination effects 7. On the other hand, natalizumab and fingolimod treatment options have been associated with patient compliance problems like safety and association with various risks in patients 9. In present practice, DMF is being prescribed in oral form, with a frequency of 2-3 times a day 2. Despite immense promises in relapsing MS, DMF has been reported to be unstable in gastrointestinal milieu and also induces gastrointestinal irritation. Other concerns like multiple dosing, compromised brain permeability, poor patient compliance and economic hurdles are the major challenges in proper utilization of DMF 6. Brain is most tightly packed organ in human body

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. Blood brain barrier (BBB), a diffusion

barrier, provides a protective barrier to brain and regulates the entry of molecules including toxins, which are harmful for the brain 11. Entry of molecules across BBB is not possible due to the tight junctions in BBB. Variety of nanotechnology based approaches are being employed now-a-days to explore the potential of neurological agents in various central nervous system disorders. It has been established that nanoparticles below 100 nm can easily cross the BBB

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. Numerous approaches like chemical modifications of marketed


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molecules, pro drugs, receptor-mediated delivery and drug efflux inhibitions have also been employed to deliver the drug to the brain 10. Lipidic carriers like solid lipid nanoparticles and nanolipidic carriers (NLCs) are also reported to easily cross BBB, due to endocytosis mechanism and lower particle size 13,14. Our aim for this current research paper was to prepare DMF-tocopherol acetate NLCs for better brain delivery. Developed DMF-tocopherol acetate NLCs were evaluated for particle size, zeta potential, PDI, TEM, caco-2 cellular permeability, in-vitro drug release studies, invivo pharmacokinetics and biodistribution studies. Literature survey revealed that no such preclinical studies for DMF delivery by means of NLCs has been attempted yet. 2.

Results and discussion

2.1 Physiochemical attributes Average particle size, zeta potential and PDI of the formulation were observed to be 69.70 ± 8.18 nm, -9.71 ± 0.80 mV and 0.317, respectively. The lower size assured better scope of brain delivery, as supported by reported literature

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. Lower PDI value indicated

homogeneity in the observed size distribution data. Developed formulation was found to be spherical in shape, as portrayed in Figure 1. TEM microphotograph confirmed smooth surface of the developed DMF-tocopherol acetate NLCs. EE and DL values were found to be 90.12 ± 1.56 % and 20.13 ± 0.56%, respectively. Better entrapment and drug loading, assured substantial drug carrying capacity of the developed nanosystem, which is one of the most desired attributes 14.



Figure 1: TEM microphotograph of DMF-tocopherol acetate NLCs 2.2 In-vitro release studies Drug release pattern from the DMF-tocopherol NLCs and plain DMF dispersion was studied using dialysis method, and the drug release profile is shown in Figure 2. It can be conferred from release studies that controlled drug release behaviour was observed from the DMFtocopherol acetate NLCs to that of pure drug. The drug release flux values for the developed DMF-tocopherol acetate NLCs and plain drug were observed to be 61.71 µg. h-1. cm-2 and 95.46 µg. h-1. cm-2, respectively. Drug release from NLCs (68% in 24 h) was significantly different from that of plain drug (~100% in 24 h; p

Enhanced Brain Delivery of Dimethyl Fumarate Employing Tocopherol

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