Preclinical Explorative Assessment of Dimethyl Fumarate-Based

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Preclinical Explorative Assessment of Dimethyl Fumarate-Based Biocompatible Nanolipoidal Carriers for the Management of Multiple Sclerosis Pramod Kumar, Gajanand Sharma, Varun Gupta, Ramanpreet Kaur, Kanika Thakur, Ruchi Malik, Anil Kumar, Naveen Kaushal, and Kaisar Raza ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00519 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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ACS Chemical Neuroscience

Preclinical Explorative Assessment of Dimethyl Fumarate-Based Biocompatible Nanolipoidal Carriers for the Management of Multiple Sclerosis Pramod Kumar1, Gajanand Sharma2, Varun Gupta3, Ramanpreet Kaur4, Kanika Thakur2, Ruchi Malik1, Anil Kumar3, Naveen Kaushal4 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

Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies (UGC-CAS), Panjab University, Chandigarh, India-160014 4

Department of Biophysics, Panjab University, Chandigarh, India-160014

.

*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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Abstract Multiple sclerosis (MS) is a neurodegenerative disease, where myelin sheath damage occurs due to internal and external factors and it affects especially young population. Dimethyl fumarate (DMF) is a promising agent for MS though associated with concerns like poor brain permeation, multiple dosing and gastro intestinal flushing. The present study attempts to evaluate preclinical performance of specially-designed DMF-based lipoidal-nanoparticles in cuprizone-induced demyelination rodent model. The studies proved the efficacy of lipid-based nanoparticles, containing DMF in once-a-day dosage regimen, over thrice-a-day plain DMF administration on the crucial parameters like motor coordination, grip strength, mortality, body weight and locomotor activity. However, neither blank lipid nor the blank neuroprotective (vitamin A, D and E) loaded nanoparticles were able to elicit any desirable behavioural response during the studies. The histopathological studies showed that the designed once-a-day DMF nanomedicines were well tolerated as well as rejuvenated myelin sheath vis-à-vis plain thrice-a-day regimen. The findings are the “proof of concept” for a biocompatible nanomedicine for MS with a huge promise of effective brain delivery and patient compliance on the grounds of reduction of dosage frequency. Keywords: SLNs, NLCs, remyelination, cuprizone, fat soluble vitamins, motor co-ordination, grip strength, neuroprotection, patient compliance


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1.

Introduction

Neurological disorders have been reported as the third largest disease burden on the globe, as per World Health Organization.1 Multiple sclerosis is an autoimmune neurological disorder affects 2.5 million population across the globe. MS destroys myelin sheath of the neurons in the central nervous system. MS is characterized by symptoms like walking disability and cognitive problems. The disease target is generally relatively young population and no gender differentiation has been reported.2 Inflammation and oxidative stress are the main agreed causes for MS, which result in demyelination of neurons, precipitating the symptoms.2–4 Various management therapies for relapsing MS are available in the market including dimethyl fumarate (DMF).5 Fumaric acid esters like DMF and monomethyl fumarate (MMF) is in practice for treatment of plaque psoriasis in Germany since 1990.6 Later on, DMF, a fumaric acid ester has been recently approved by various federal agencies for the oral management of MS. DMF is a drug which isn’t discovered with screening and modern optimization methods. DMF long term safety data has been seen in Europe since two decades with anti-inflammatory responses.7–9 In various studies, it has been shown that DMF is rapidly metabolized in its main metabolite i.e. MMF by esterase enzyme present in the gastrointestinal tract.10 DMF enjoys a special status amongst various disease-modifying drugs (DMDs) for MS and has emerged as a promising treatment option for relapsing–remitting MS, supplemented with anti-inflammatory and neuroprotective actions apart from the ease of oral administration.11,12 Most of the currently available DMDs for MS do not remain relapse-free and generally require parenteral administration. Apart from these concerns, the efficacy of these DMDs has been reported to be “partial”.12 DMF seems effective still its mechanism of action is less understood, but multiple pathways like nuclear factor (erythroid derived 2)-like2 (NRF2) and Kelch-like erythroid cell-derived (ECH) associated protein-1 (KEAP-1) are believed to be involved for execution of its action.13 However, being orally effective, DMF also offers huge scope for research owing to the associated concerns like gastrointestinal stability, high dose and frequency of administration, lower brain permeability, economic hurdles and poor patient compliance.14–16 Recently, our research group has generated evidences for better oral delivery prospects of this drug using variety of lipid-based nanoparticles incorporating neuroprotective excepients like fat soluble vitamins.17–22,23,24,25 Till date



physicochemical, stability, drug release, cellular uptake and pharmacokinetic evidences along with biodistribution studies have been published for once-a-day nanoformulations of DMF employing neuroprotectives like tocopherol acetate, cholecalciferol and retinol acetate.14–16 However, no studies have been reported on the pharmacological outcomes of the designed nanomedicines in appropriate animal models like cuprizone-induced demyelination mouse model.26–28 The present preclinical evaluation observes the effect of the designed nanosystems of DMF on the body weight, locomotor activity, motor coordination and functional observation battery performance of mice receiving cuprizone, a demyelinating agent. At the end of the study, histopathological evaluation of brain and stomach of each mouse was performed to infer the myelination status and gastric mucosa damage, respectively. 2.

Results and Discussion

2.1.

Physiochemical attributes of the nanoparticles

Developed formulations have been characterized for various material aspects like average particle size, zeta potential, polydispersity index and entrapment efficiency. The average particle size of developed nanocarriers ranged between 69.70 ± 8.18 nm to 198.70 ± 5.96 nm. The values of PDI ranged between “0.317 to 0.608”. Meanwhile, all the nanoparticles offered zeta-potential values in the range of -1.57 ± 0.03 mV to -9.71 ± 0.81 mV. All the developed systems offered EE > 85%, indicating a better selection of drug to lipid ratio. The same can be traced in detail in previous research papers published by the same group.14–16 2.2.

Body weight

Figure 1 depicts the variation in average body mass of the animals of various groups, receiving the designated treatments. Cuprizone treatment group was observed for the maximum reduced body weight within a month vis-a-vis the group receiving saline or vehicle. Subsequent reduction in body weights were also observed in groups receiving cuprizone only, pure DMF and blank tocopherol acetate, blank cholecalciferol, blank retinol acetate by 8.33%, 5.71%, 6.45%, 7.14% and 8.33 to that of first day of the treatment. Enhancement in the body weight was observed in the groups receiving vehicle, blank SLNs, DMF-O-SLNs, DMF-tocopherol SLNs, DMFcholecalciferol SLNs and DMF-retinol acetate SLNs, by 2.94%, 3.45%, 2.27%, 5.41%, 1.67% and 4%, respectively. It can be vouched from the studies that DMF treatments with vitamins slightly increased the body weight. It might be due to solid lipid used in the preparation of SLNs.29 In cuprizone only group, 16.67% mortality was observed, however, no deaths were


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# $ *

5

$ *

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Cuprizone + blank cholecalciferol SLNs

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-5

-10

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* Group type

2.3.

Cuprizone + blank retinol acetate SLNs

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Figure 1:

Cuprizone + DMF-retinol acetate SLNs

Cuprizone + blank tocopherol acetate SLNs

Cuprizone + DMF-tocopherol acetate NLCS

Cuprizone + Blank SLNs

Cuprizone + DMF-O-SLNs

Cuprizone + pure dmf

Cuprizone

10

Vehicle

observed in other animal groups. The studies provided an inference that the administration of the designed nanomedicines did not alter normal metabolism rate of the animals, whereas the oral administration of the plain drug rather resulted in decreased weight of the animals. The findings when correlated with other measurable outcomes provide a promising treatment for this orally prescribed drug for MS.

% change in body weight

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


* $

* $

Graphical representation of percentage change in body weight in different group (*, $ and # indicate the significant difference from the saline group, cuprizone only group and cuprizone and pure DMF treated group, respectively at p < 0.001)

Assessments of behavioural studies

2.2.1. Locomotor activity Locomotor activity was recorded in different groups using actophotometer and the results have been presented in Figure 2. These findings suggest that cuprizone-treated group showed the maximum deviation from the normal locomotor function on Day 30 to that of Day 1, followed by the groups receiving blank formulations. However, the groups receiving the designed DMFloaded nanocarriers offered better locomotor activity to the group receiving plain DMF thrice-a-



day, though the outcomes from the DMF-loaded tocopherol acetate NLCs were the best, as close to the saline receiving group. The studies clearly demonstrated the aptness of the model, as demyelination (as in MS) is characterized by suppression in locomotion30, meanwhile the administration of the treatments resulted in improvement of the same over a period of 1 month. The developed DMF-loaded nanocarriers clearly demonstrated the supremacy to thrice-a-day plain DMF, that too in once-a-day equivalent dose, indicating the huge promise in improving the performance and pharmacological outcome of the DMF therapy in conditions like MS.

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Cuprizone + DMF-cholecalciferol SLNs




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$ *

60

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Group type

Figure 2: Graphical representation of variation in the locomotor activity to that in the Day 1(*, $ and # indicate the significant difference from the saline group, cuprizone only group and pure DMF treated group, respectively, at p < 0.001) 2.2.2. Motor co-ordination Motor coordination, balance on rod and grip strength were determined by rota-rod test. All animals were observed to increase their motor coordination and grip strength during training period. It was observed that there was not more than 6% change in all groups on Day 1, as shown


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in Figure 3. It was also observed that there was no significant difference in the readings of the vehicle treated group on 30th day vis-a-vis Day 1. Cuprizone treated group showed the least motor coordination and balance on Day 30 to that of Day 1. Highest motor coordination and balance (90.60% change) was observed in the group receiving DMF-tocopherol acetate NLCs to that of all treated groups.

*


Cuprizone + DMFcholecalciferol SLNs


# $ *

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Locomotor activity count/5 minute (% vehicle control group)

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Group type

Figure 3: Graphical representation of percentage variation in activity (fall in seconds) (*, $ and # indicate the significant difference from the saline group, cuprizone only group and pure DMF group, respectively, at p < 0.001) 2.2.3. Functional observation battery test No abnormal behaviour like seizures and weakness was observed in the animals of vehicle treated group. Seizures and weakness were observed in cuprizone treated group and blank SLNs group pre-treated with cuprizone 30th day. Weakness and normal activities were reduced in the cuprizone treated group. 2.4.

Histopathological evaluation of brain and stomach

Harvested brain samples from each group were stained with luxol fast blue dye for visibility of the blue stained myelin sheath. The corpus callosum region which is reported to show maximum



demyelination in the selected model was observed, as suggested by literature.31 The microphotographs of the saline treated group showed significant myelination on Day 1 as well as Day 30, which was taken as the reference of 100% for comparison. On the other hand, approx. 42.72% demyelination was observed in cuprizone only treated group on 30th day. As shown in Figure 4, 27.95% demyelination was observed in the group receiving pure DMF (thrice-a-day) the observed value for DMF-tocopherol acetate NLCs group was only 10.94%. It can be vouched from the histopathological studies that DMF-tocopherol acetate NLCs was more effective on myelin sheath recovery to that of other treated groups; however, all the DMF-loaded nanocarriers were observed to be better than plain DMF. The findings were in consonance with behavioural studies, vouching the superiority of vitamin E-based NLCs over other lipid-based nanocarriers. The microphotographs of the stained brain sections have been presented as Figure 5. The findings are in close agreement with the published evidences of enhanced brain delivery by the lipid-based nanocarriers, testifying the fact of enhanced brain delivery and subsequently re-

100

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Cuprizone + blank cho lec alci ferol SLNs

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Cuprizone + DM F-tocopherol ac etate NLCS

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myelination of neurons on diseases like MS.14–16

% chnage in myleination

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Figure 4: Graphical representation of percent change in myelination in the groups receiving various treatments (*, $ and # indicate the significant difference from the saline group, cuprizone only group and pure DMF group, respectively, at p < 0.001)




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Figure 5: Histopathological investigations of the brain slides of animals receiving the treatments of: (A) Saline; (B) Cuprizone; (C) Cuprizone + pure DMF; (D) Cuprizone + DMF-O-SLNs; (E) Cuprizone + blank SLNs; (F) Cuprizone +DMF-tocopherol acetate NLCs; (G) Cuprizone + blank tocopherol acetate NLCs; (H) Cuprizone + DMF-retinol acetate SLNs; (I) Cuprizone + blank retinol acetate SLNs; (J) Cuprizone + DMF-cholecalciferol SLNs; (K) Cuprizone + blank cholecalciferol SLNs Stomach slides were stained with eosin and haematoxylin and the respective microphotographs have been shown in Figure 6. Substantial mucosal damage was observed in the cuprizone-treated group, followed by the group receiving plain DMF. There were no evidences of granuloma or malignancy in the stomach samples of the animals receiving the designated treatments. The findings are important key in establishing the safety profile of a drug which is known to induce gastric irritation in subchronic assessment of one month. It also provides an evidence of oral tolerance of the developed lipid-based nanosystems.




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Figure 6: Pictorial representation of the histology of the stomach of animals receiving the treatments (A) Saline; (B) Cuprizone; (C) Cuprizone + pure DMF; (D) Cuprizone + DMF-OSLNs; (E) Cuprizone + blank SLNs; (F) Cuprizone +DMF-tocopherol acetate NLCs; (G) Cuprizone + blank tocopherol acetate NLCs; (H) Cuprizone + DMF-retinol acetate SLNs; (I) Cuprizone + blank retinol acetate SLNs; (J) Cuprizone + DMF-cholecalciferol SLNs and (K) Cuprizone + blank cholecalciferol SLNs 3.

Conclusions

Data obtained from the preclinical assessment of the pure DMF, DMF-loaded lipid nanoparticles along with tocopherol acetate, cholecalciferol and retinol acetate and respective blank formulations established the proof-of-concept for once-a-day preclinical dosage regimen for DMF in MS that too with enhanced efficacy and biocompatibility. Interestingly, the subchronic preclinical protocol also gave an inference that naive vitamins were not so useful in MS like condition; however, the outcomes were synergistically improved in conjunction with DMF. Outcomes of such holistic studies can be envisioned as a ray of hope for better therapeutic options for numerous neurological disorders, where the delivery of drug to brain is a major hurdle. 4.

Materials

DMF and MMF were purchased from M/s Sigma-Aldrich, Banglore, India. Cuprizone was bought from M/s Alfa Asser Limited, New Delhi. Methanol, water and acetonitrile were bought from M/s Spectrochem Pvt. Limited, Mumbai, India. Picric acid was delivered by M/s Fisher Scientific India Pvt. Limited, Mumbai, India. Ethanol was purchased from M/s Jai Chemicals and Pharma Works, Jaipur, India. Oyster BDS premium C18 (250 X 4.6, 5µm; Batch no. 43/053) HPLC column was purchased from M/s Merck Specialities Pvt. Ltd., Mumbai, India. Luxol fast blue dye was supplied by the M/s/ Sigma Aldrich Corporation, New Delhi, India. All the reagents and chemicals were used of analytical grade and used as such. 5.

Methods

5.1.

Physiochemical attributes of the nanoparticles


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Hot micro emulsification technique was used to formulate DMF-O-SLNs/DMF-tocopherol acetate NLCs/DMF-retinol acetate SLNs/DMF-cholecalciferol SLNs. Developed nanocarriers have been well characterized by particle size, zeta potential, polydispersity index, transmission electron microscopy. Entrapment efficiency and drug loading have been determined using dialysis bag method. The same has been reported in detail in previous research papers published by the same group.14–16 5.2.

Preclinical assessments

Healthy Laca mice (25-50 g) were used in the whole studies at Central Animal House facility of Panjab University, Chandigarh. All animals were kept in dark and light standard conditions in ventilated cages with food and water ad libitum. All the healthy Laca mice were acclimatized in laboratory conditions before experimentation. All the experiments including training session were performed between 10:00:00 to 17:00:00 and were duly approved by the Institutional Animal Ethics Committee, Panjab University, Chandigarh. Animal marking, drug and treatment schedule Picric acid (1%) solution was used to mark the Laca mice groups in the standard manner. All the designated formulations were orally administered once-a-day, unless stated.

Detail list of

various groups and dosed have been mentioned in Table 1. The details of the formulations used for the preclinical studies have already been published.14,15 Table 1: Details of the animal groups and respective oral dose administration regimen S. No.

Group (n= 06)

Group Details

Dose (DMF equivalent to 3 mg/kg-orally)

1.

Group 1

Vehicle or normal saline

Equivalent volume once-aday

2.

Group 2

Cuprizone suspension

6 mg/day once-aday

3.

Group 3

Pure DMF

3 mg/kg thrice-aday

4.

Group 4

DMF-optimized

Solid


lipid 3 mg/kg once-a-


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nanoparticles (DMF-O-SLNs)

day

5.

Group 5

SLNs Blank

3 mg/kg once-aday

6.

Group 6

DMF-tocopherol acetate NLCs

3 mg/kg once-aday

7.

Group 7

Blank-tocopherol acetate NLCs

3 mg/kg once-aday

8.

Group 8

DMF-retinol acetate SLNs

3 mg/kg once-aday

9.

Group 9

Blank-retinol acetate SLNs

3 mg/kg once-aday

10.

Group 10

DMF-cholecalciferol based SLNs

3 mg/kg once-aday

11.

Group 11

Blank-cholecalciferol SLNs

3 mg/kg once-aday

All the formulations were administered for 30 days the the respective treatment groups. In all the groups (except Group 1), cuprizone suspension (6mg/kg), once-a-day, was administered orally, 1 hour prior to the prescribed dosing. Selection of dose has been based on the available literature reports.31,32 All the animals were observed for the parameters (listed in subsequent sections) on weekly basis. 5.1.1. Body weight Body weights of the Laca mice were measured on the first day and every week, during the whole experimentation protocols. Percentage change in body weight was reported with respect to the average weight on the day 1. Percentage change in body weight was calculated as: Percentage change in body weight = Body weight [(Day 30 - Day 01) X 100] 5.1.2. Behavioural assessments Behavioural studies were performed employing actophotometer, rota-rod test and functional observation battery test. Each Laca mouse was acclimatized and habituated for the


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actophotometer, rota rod test and functional observation battery test in the laboratory, as per the set protocols.33 5.1.2.1.

Locomotor Activity

Actophotometer (IMCORP, Ambala, India) was used to monitor the locomotor activity of the mice receiving various treatments. The motor performance of the Laca-mice was detected using infra-red beams above the floor of testing area. Animals were habituated prior to experiment for 03 minutes before making actual experiment. Observation in the actophotometer was recorded for 05 minutes and reported as the count per 05 min.33 5.1.2.2.

Motor-coordination

Motor-in-coordination and grip strength of the animals were evaluated using rota-rod apparatus (IMCORP, Ambala, India). All animals were trained on rota rod during training sessions, before dosing. Cut off time was fixed as 120 seconds and three trials were performed after 05 minutes gap. Animals were placed on rotating rod with speed 25 rpm. Average fall of time was recorded and expressed as the average counts per 120 sec.27 5.1.2.3.

Functional observation battery test

Functional observation battery test was employed to access the neurotoxic effects in Laca mice. In the current study, an experimental box (box size 40 × 60 cm) was used to access the abnormal behaviour like seizures and weakness. Each animal was placed in a box for 05 minutes, before experiment, as a training session for acclimatization. Abnormal responses like (seizures, weakness, tremors, ataxia and convulsions) were recorded in five minutes’ time intervals.27,31 5.3.

Histopathological evaluation of brain and stomach

At the end of the studies, brain and stomach from the animals of each group were harvested. Isolated brain and stomach samples were stored in formalin solution till further processing. Microtome was used to make the slices of the tissue. For histopathological evaluations, the brain sections were stained with 1% luxol fast blue ethanolic solution and the stomach sections were stained with eosin and haematoxylin.26,28 The microphotographs were clicked at suitable magnification and inferences were drawn accordingly.



6.

Author information

Author contributions Mr Pramod Kumar designed, executed the experiments and prepared the manuscript; Prof. Anil Kumar, Mr. Gajanand Sharma, Ms. Kanika Thakur and Mr. Varun Gupta helped in pharmacological studies; Dr. Naveen Kaushal and Ms. Ramanpreet Kaur supported in histopathological evaluation; Dr Kaisar Raza and Dr Ruchi Malik supervised in hypothesis building, experimentation and final drafting of the manuscript, and also arranged the chemicals and facilities, required for the experimentation. Acknowledgements Authors would like to acknowledge Dr. Lal PathLabs, Tilak Nagar, Jaipur to help in interpretation of histological images of stomach. Conflict of interest Authors report none conflict of interest. Funding sources The support in the form of senior research fellowship and partial contingent grant from University Grants Commission (UGC), New Delhi, India to Mr. Pramod Kumar (F./201415/NFO-2014-15-OBC-RAJ-8108/(SA-III/Website) is acknowledged. 7.

References

(1) WHO | WHO Mortality Database. WHO. [Last accessed on 07/01/2018] (2) Singhal, B. S. (1985) Multiple sclerosis--Indian experience. Ann. Acad. Med. Singapore 14, 32–36. (3) Singhal, B. S., and Advani, H. (2015) Multiple sclerosis in India: An overview. Ann. Indian Acad. Neurol. 18, S2–S5. (4) Loma, I., and Heyman, R. (2011) Multiple Sclerosis: Pathogenesis and Treatment. Curr. Neuropharmacol. 9, 409–416.


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Page 22 of 22

A Table of Contents Graphic (TOC) Manuscript

title:

Preclinical

Explorative

Assessment

of

Dimethyl

Fumarate-Based

Biocompatible Nanolipoidal Carriers for the Management of Multiple Sclerosis Authors: Pramod Kumar, Gajanand Sharma, Varun Gupta, Ramanpreet Kaur, Kanika Thakur, Ruchi Malik, Anil Kumar, Naveen Kaushal and Kaisar Raza


Preclinical Explorative Assessment of Dimethyl Fumarate-Based

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