(T1430-02-10) Evaluating Treatment Efficiency of RV and Kinase Inhibitor (KI) in Combination for Alzheimer’s disease: Cell Line Study

Purpose: Alzheimer’s disease (AD) is a neurodegenerative disorder with a slow progression and devastating outcomes. AD accounts for 60% to 80% estimated cases of dementia. Presently, the treatment of AD using single drug has proven to be insufficient due to the complexity associated with AD. So, it can be well interpreted that combination of drugs with different mechanism can be better treatment strategy for AD. RV, a Food and Drug Administration (FDA) approved drug has been a drug of choice for AD due to its property of inhibiting both acetylcholinesterase (AChE) and butyrylcholinesterase enzyme (BuChE). Another drug, KI has ability to restores memory function by preventing the degeneration of dopaminergic neuron in AD. Since both the drugs have beneficial effect against AD therefore, their deposition at target site must be in desired amount to achieve the therapeutic effect. But both the drugs possess various limitations such poor oral bioavailability, low solubility, and permeability. Therefore, to overcome the associated problems and to make them available in the brain in significant concentration, this combination is required to be loaded in nano formulation followed by administration via nasal route. This study emphases on the delivery of the RV and KI in combination to brain to achieve their combined synergistic effects against AD. This ratio of combination has been selected in such a way that it makes neurons cells to survive more rather than getting killed due to toxicity of the drugs. Thus, a cell line study was conducted to optimize the ratio of RV:KI that could be loaded in the nano formulation followed by delivery to the brain via nasal route. As, it would help in the management of AD without facing any side effects and loss in the dose of the drug.

Methods: The selection of dose of the RV and KI used for the management of AD was carried out by performing cell lines study. Neuro-2a cells lines which are rapidly growing mouse neuroblastoma cells were used. The treatment of these cells was done with different concentration of individual drug ranging from 0.97 to 2000 µg/ml for 24h. The viability of these cells was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Later, the IC₅₀ value of RV and KI were determined followed by the optimization of the ratio of both the drugs by combination index (CI) based on the effect of them on the viability of Neuro-2a cell. Moreover, this ratio was selected by keeping the concentration of RV constant, followed by increase in the concentration of KI.

Results: MTT assay was performed on Neuro-2a cells that were treated with different concentration of each RV and KI for 24h in which it was observed that there was dose dependent % cell viability of each drug. On increasing the concentration of RV and KI individually from 0.97 to 2000 µg/ml, the mean % cell viability was observed to be 68.42 ± 0.41 to 36.63 ± 0.34 whereas, in KI it was observed to be 70.85 ± 0.44 to 36.51 ± 0.37. On obtaining the % cell viability data, the IC₅₀ value of RV and KI was calculated and found to be 125 µg/ml and 62.5 µg/ml respectively. Based on the IC₅₀ of RV and KI the CI study was conducted by taking different ratios starting from 1:1 to 1:7 (RV: KI), the fraction affected (Fa) was found to be 0.3648 to 0.1872, whereas CI was observed to be 0.106 to 0.004 respectively.

Conclusion: Based on the cell line study conducted on Neuro-2a cells lines, data suggested that the 1:1 ratio of RV and KI for drug combination, have the greatest possible synergistic effect and same has been observed based on the dose effect curve and combination index plot. So, this ratio could be taken up further for the development of the nano formulation for the treatment of AD.

References: 1) Savage MJ, Gingrich DE. Advances in the development of kinase inhibitor therapeutics for Alzheimer's disease. Drug Development Research. 2009 Mar;70(2):125-44.
2) Haider MF, Khan S, Gaba B, Alam T, Baboota S, Ali J, Ali A. Optimization of RV nanoemulsion for enhanced brain delivery: In-vivo and toxicity evaluation. Journal of molecular liquids. 2018 Apr 1; 255:384-96.
3) Erdő F, Bors LA, Farkas D, Bajza Á, Gizurarson S. Evaluation of intranasal delivery route of drug administration for brain targeting. Brain Res Bull. 2018 Oct; 143:155-170. doi: 10.1016/j.brainresbull.2018.10.009. Epub 2018 Oct 25. PMID: 30449731.

Acknowledgements: The authors are thankful for DST-FIST grant to Department of Pharmaceutics, School of Pharmaceutical Education & Research, Jamia Hamdard, New Delhi, India.

Graphical representation of mechanism of action of drugs for the treatment of AD