{"id":58450,"date":"2024-06-25T11:26:49","date_gmt":"2024-06-25T11:26:49","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58450"},"modified":"2024-07-03T17:03:15","modified_gmt":"2024-07-03T17:03:15","slug":"pharmacokinetic-and-safety-evaluation-of-mbzm-n-ibt-a-lead-against-chikungunya-virus","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/pharmacokinetic-and-safety-evaluation-of-mbzm-n-ibt-a-lead-against-chikungunya-virus\/","title":{"rendered":"Pharmacokinetic and Safety Evaluation of MBZM-N-IBT, A Lead Against Chikungunya Virus"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Infection by Chikungunya virus (CHIKV) is categorised by WHO as a “neglected tropical disease” (WHO, 2014)1<\/sup>. Although it has a tropical origin, it is no longer limited to tropical areas. Gradually, it has become a global pathogen in many countries with different climatic conditions2,3<\/sup>. Although no specific antiviral is approved for its management, there have been continued efforts to identify potential drug candidates for further development as antiviral for CHIKV4-9<\/sup>. Our group recently patented MBZM-N-IBT (Figure 1) for its in vitro<\/em> effects against CHIKV infection (Indian Patent No. 347450). Chemically it is 1-[(2-methyl benzimidazole-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea10<\/sup>. Unlike the prior art in its chemical class (methisazone), it was reported as a potent inhibitor of CHIKV10<\/sup>.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Structure of MBZM-N-IBT<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In addition to anti-CHIKV properties, it was shown as a potent inhibitor of the Herpes Simplex Virus (HSV1) in vitro<\/em>11<\/sup>. The effects against both RNA (CHIKV) and DNA (HSV) virus suggest its broad spectrum of action and potential for further progress as an antiviral candidate. Pharmacokinetic study plays a vital role in drug development as it helps in the prediction of efficacy and toxicity-related events. Thus, to better understand the toxicity and the effectiveness of MBZM-N-IBT in vivo, <\/em>it is necessary to assess its pharmacokinetics through a rapid, simple, and effective bio-analytical method12<\/sup>. MBZM-N-IBT is a chemical substance and may cause acute toxicity within 24 hours following single-dose administration. When continuously administered, it could lead to chronic toxicity in the living system when used over a more extended period of time.13,14<\/sup>. A key criterion to assess acute toxicity is the determination of the lethal dose (LD50), which is defined as the dose that kills 50% of the population of test animals.<\/p>\n\n\n\n

Furthermore,\nit appears as a guiding dose for in vivo<\/em>\nefficacy and toxicity studies15<\/sup>. Accordingly, the LD50 <\/sub>was estimated in the present study before assessing the chronic toxicity. In silico<\/em> models\nhave been widely used during the toxicology analysis16-18<\/sup>\nto reduce safety-related attrition in drug development. So, the metabolites of MBZM-N-IBT were predicted through in silico<\/em> models, and their potential\ntoxicity was assessed to support the findings\nof in vivo<\/em> toxicology. With these thoughts in mind, the goal of the\ncurrent investigation was to evaluate both the toxicity and pharmacokinetics of\nMBZM-N-IBT to assess its potential for further progress as an antiviral\ncandidate.  <\/p>\n\n\n\n

Material and Methods<\/strong><\/p>\n\n\n\n

Chemicals and Reagents <\/strong><\/p>\n\n\n\n

MBZM-N-IBT was synthesized and purified by column chromatography following our established protocol10<\/sup>. The purity (\u02c398%) was monitored by HPLC. Metformin, Metronidazole, Ranitidine, Paracetamol, Phenobarbital, Cefixime, Salicylic acid, Cetrizine, omeprazole, and Losartan were all obtained from industry with high purity (\u02c395%). The HPLC-grade solvents, such as acetonitrile, water, and methanol, have been purchased from Merck, India. Drugs\/test compounds were added to 1% carboxy methyl cellulose (CMC) in distilled water at room temperature, and fresh crude suspensions were prepared. <\/p>\n\n\n\n

Animals and Ethical Statement<\/strong><\/p>\n\n\n\n

The Institutional Animal Ethics Committee (IAEC) of the School of Pharmaceutical Sciences of Siksha O Anusandhan (Deemed to be University), Bhubaneswar, India, (Reg. no. 1171\/PO\/Re\/S\/08\/ CPCSEA) granted its approval to the experimental protocol. The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) approved all experiments and supervised the way they were conducted. To evaluate acute and chronic toxicity, healthy Wistar albino rats of either sex, around 150-200g, were used. They were kept in polyacrylic cages with 12-hour light\/dark cycles at optimal temperatures of 25\u00b12\u00b0C and 60\u00b15% relative humidity. The animals had complete access to commercially available food and water. <\/p>\n\n\n\n

Animal grouping and husbandry<\/strong><\/p>\n\n\n\n

Animals were grouped into subgroups according to the experiments after seven days of acclimation. For the acute toxicity study, animals were divided into 4 groups arbitrarily (n=3, female rats). For the chronic toxicity study, animals (both male and female rats) were randomly divided according to the dose and the time limit of the experiment. The in vivo<\/em> pharmacokinetic study was performed by taking albino rats at each time point (n=6). All of the animals were housed in polypropylene cages with stainless steel covers that contained water bottles and feeders.<\/p>\n\n\n\n

Dosing<\/strong><\/p>\n\n\n\n

MBZM-N-IBT was mixed with 1% carboxy methyl cellulose to achieve the desired concentration. Dosing formulations were stored at 4\u00baC. The formulations were brought to room temperature and thoroughly mixed before administration.<\/p>\n\n\n\n

Acute oral toxicity of MBZM-N-IBT<\/strong><\/p>\n\n\n\n

Following the OECD-423 guidelines for testing chemicals (OECD, 2000)19,20<\/sup>, this experiment was carried out to estimate the LD50 of MBZM-N-IBT. Following an overnight fast of approximately 15 to 16 hours, all the animals were given a single oral dose of MBZM-N-IBT with unrestricted access to water. The dose volume was 10 mL\/kg. The starting dose was 50 mg\/kg body weight. The animals were next examined with 300 mg\/kg and 2000 mg\/kg single oral doses. The toxic impact of each and every experimental animal was observed for the entire day. For the first 4h of daily administration, animals were critically monitored for any signs of toxicity. The changes in behavior, lethargy, appearance, salivation, illness, body weight, and mortality were examined during this period. The animals were further observed for 14 days.<\/p>\n\n\n\n

Chronic oral toxicity of MBZM-N-IBT<\/strong><\/p>\n\n\n\n

Chronic toxicity was conducted as per the OECD-452 guidelines (OECD, 2008)21<\/sup>. As per the recommendations of this guideline, the highest dose that can be administered is 1000 mg\/kg. The animals were divided into 4 groups arbitrarily. Each group contained male and female rats (n=20 for each sex). The Group-I (normal control) rats received 1% CMC. Group II, III, and IV rats received oral MBZM-N-IBT at 1000 mg\/kg, 500 mg\/kg, and 50 mg\/kg doses. All doses were administered once per day with a fixed schedule. Animals were observed for gross behavioral, neurological, and autonomic effects. Also, changes in eyes, skin\/fur, salivation, sleep, diarrhea, coma, and mortality were noted twice daily throughout the testing period. On a weekly basis, the rats’ body weight was monitored. After the completion of the period, ketamine was injected intraperitoneally, and rats were euthanized. For histological analysis, vital organs like the liver, heart, lung, and kidney were carefully excised, weighed, and preserved in 10% buffered formalin.<\/p>\n\n\n\n

Bioanalytical method development and Pharmacokinetics of MBZM-N-IBT<\/strong><\/p>\n\n\n\n

A\nbinary gradient HPLC (Shimadzu, Japan)\nwas employed, along with an LC-20AD pump and SPD-M20A prominence diode array\ndetector. Samples were injected into a KROMASIL PROCHROME C18 analytical column\n(4.6 \u00d7 150mm, 5\u03bcm) using a Hamilton SYR 25\u03bcL syringe after being filtered\nthrough a syringe-driven nylon membrane filter (0.22\u00b5, 13mm). With an isocratic\nflow rate of 1.0mL\/min and water methanol (50: 50 v\/v) as the mobile phase, the\nestimation of MBZM-N-IBT was carried out. The mobile phase was degassed in an\nultrasonic bath after being filtered through a Millipore vacuum filter system\nwith a 0.45\u03bcm membrane filter. Each injection ran for ten minutes. At 360nm,\nthe analytes were observed. At ambient temperature, all experiments were\ncarried out. Following ICH guidelines Q2 (R1), a reversed-phase HPLC\nanalytical technique was developed and validated for the quantitative determination\nof MBZM-N-IBT from bulk22<\/sup>. The linearity, accuracy, precision, LOD,\nand LOQ were determined. Again, this method was verified and re-validated for\nassay from rat plasma matrix following USFDA guidelines for bio-analytical\nmethod validation23,24<\/sup>.The calibration curve was used to estimate MBZM-N-IBT in\nplasma. The in vivo<\/em>\nPharmacokinetic study was performed by taking three albino rats at each time\npoint (n=6). Animals were orally administered with MBZM-N-IBT 50 mg\/kg body\nweight. At appropriate time points (0.5, 1, 1.5, 2, 4, 8, 12, 18, and 24 hr), the\nanimals were euthanized under light ether anesthesia, and blood was obtained\nthrough the cardiac puncture, which was then collected into a pre-treated vial\ncontaining EDTA. Following blood collection, the samples were centrifuged at\n15,000 rpm for 15 minutes at 4\u00b0C to separate the plasma. Then, clear\nsupernatants were collected and subjected to deproteinization (Acetonitrile). <\/p>\n\n\n\n

The collected samples were preserved at\n-80\u00b0C until they were analyzed. Different pharmacokinetic parameters have been\ndetermined, including AUC, AUMC, MRT, Cmax<\/sub>, Tmax<\/sub>, and T1\/2<\/sub>.<\/p>\n\n\n\n

Protein binding study<\/strong><\/p>\n\n\n\n

Plasma protein binding (PPB) was\nevaluated using a modified version of a previously reported procedure25<\/sup>. Drugs with reported PPB values\nranging from 0% to 99% were selected for the research. Metformin,\nmetronidazole, ranitidine, paracetamol, phenobarbital,\ncefixime, salicylic acid, cetirizine, omeprazole,\nand losartan were all obtained from industry with high purity (\u02c395%). An\nisocratic mobile phase, composed of a 0.9% sodium chloride solution at pH 7.0,\nwas employed, and it was run at a flow rate of 0.5 mL\/min. The study was\nperformed using a chiral pack HSA column (150\u00d74 mm, 5\u00b5m). A significant\ncorrelation was achieved by generating a graph that compared the derived\ncapacity factors with the reported (known) PPB percentages.         \n<\/p>\n\n\n\n

In silico analysis of metabolites and their LD50<\/strong><\/p>\n\n\n\n

The OECD\nQSAR Toolbox, owned by the OECD and the European Chemical Agency, is a tool\nused for the assessment of chemicals and the mechanistic basis of their\ntoxicology26<\/sup>. The classical interface of QSAR tool box4.6, 2023 was\nused for the prediction of metabolite and their toxicity. MBZM-N-IBT structure\nwas used as input. Several\nmetabolism simulators were used to predict the metabolites, including in vivo\nRat metabolism, autoxidation, and hydrolysis (acidic, basic, and neutral).\nThese metabolites were utilized as inputs for predicting acute toxicity using\nthe OECD QSAR toolbox, which aids in categorizing chemicals into specific\ngroups.<\/p>\n\n\n\n

Statistics<\/strong><\/p>\n\n\n\n

All data are expressed as mean\u00b1SEM (Standard Error\nMean) and were analyzed by the XLSTAT software using one-way ANOVA followed by\nBonferroni multiple comparison tests (p\u02c20.001).<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

MBZM-N-IBT showed no acute oral toxicity<\/strong><\/p>\n\n\n\n

After administration of different single oral doses (50 mg\/kg, 300 mg\/kg, and 2000 mg\/kg) of MBZM-N-IBT, the animals were observed for the first 4h and daily up to 14 days. There were no behavioral changes or mortality (Table 1) with different doses of MBZM-N-IBT. As a result, it was classified as “Category 5” or “unclassified” as per the Globally Harmonized System (GHS) for chemical classification. Accordingly, 5000 mg\/kg was considered as the LD50<\/sub>. <\/p>\n\n\n\n

Table 1: Acute oral toxicity observation table<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Step<\/strong><\/p>\n<\/td>\n

\n

Dose<\/strong><\/p>\n

(mg\/kg)<\/strong><\/p>\n<\/td>\n

\n

Number of<\/strong><\/p>\n

Animals<\/strong><\/p>\n<\/td>\n

\n

Number of moribund\/<\/strong><\/p>\n

deceased Animals<\/strong><\/p>\n<\/td>\n

\n

Subsequent<\/strong><\/p>\n

dead animal<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

50<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

50<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

300<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

300<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

2000<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

2000<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

No significant chronic toxicity was found on chronic use of MBZM-N-IBT (50 mg\/kg) <\/strong><\/p>\n\n\n\n

MBZM-N-IBT (50 mg\/kg) did not affect the food and water intake of animals<\/strong><\/p>\n\n\n\n

Physiological\nchanges (Table 2) were observed following the administration of MBZM-N-IBT at a\ndose of 1000 mg\/kg\/day (the highest permissible dose) and 500 mg\/kg\/day. Rats\ntreated daily with 500 mg\/kg showed no abnormality for up to 28 days (Table 2).\nBeyond 42 days, their food\/water intake capacity was observed to decrease (Figure\n2). A simultaneous decrease in body weight was also observed, and it became\nsignificant after 60 days. Rats treated with 50 mg\/kg\/day showed no abnormality\neven after 120 days of administration (Table 2). The average food intake and water consumption (Figure 2) were\nobserved to be average at this dose. Although after 270 days, most of\nthe parameters were normal (Table 2), It exhibited mild sedation, along with an\n11% reduction in body weight. The colour and consistency of the stool were\nyellowish and loose. The dose at 1000mg\/kg\/day showed toxicity (Figure 3) in\nrats after 28 days of continuous administration. Since mortality was found with\nthe highest dose, this was discontinued.<\/p>\n\n\n\n

Table 2: Behavioural changes in the animal during chronic toxicity studies following treatment with MBZM-N-IBT<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Parameters<\/strong><\/p>\n<\/td>\n

\n

1000 mg\/kg<\/strong><\/p>\n<\/td>\n

\n

500 mg\/kg<\/strong><\/p>\n<\/td>\n

\n

50 mg\/kg<\/strong><\/p>\n<\/td>\n

\n

50 mg\/kg<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

28 days<\/strong><\/p>\n<\/td>\n

\n

60 days<\/strong><\/p>\n<\/td>\n

\n

120days<\/strong><\/p>\n<\/td>\n

\n

270 days<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Condition of fur<\/p>\n<\/td>\n

\n

Abnormal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n<\/tr>\n

\n

Skin<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n<\/tr>\n

\n

Subcutaneous swelling<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n<\/tr>\n

\n

Eyes dullness<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n<\/tr>\n

\n

Breathing abnormalities<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n

\n

Nil<\/p>\n<\/td>\n<\/tr>\n

\n

Food intake<\/p>\n<\/td>\n

\n

Decreased after 7days<\/p>\n<\/td>\n

\n

Normal till 28 days<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n<\/tr>\n

\n

Colour\/consistency of feces<\/p>\n<\/td>\n

\n

Abnormal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Yellowish\/loose<\/p>\n<\/td>\n<\/tr>\n

\n

Alertness<\/p>\n<\/td>\n

\n

Abnormal after one week<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n<\/tr>\n

\n

Convulsions<\/p>\n<\/td>\n

\n

Not observed<\/p>\n<\/td>\n

\n

Not observed<\/p>\n<\/td>\n

\n

Not observed<\/p>\n<\/td>\n

\n

Not observed<\/p>\n<\/td>\n<\/tr>\n

\n

CNS<\/p>\n<\/td>\n

\n

Sedation<\/p>\n<\/td>\n

\n

Sedation<\/p>\n<\/td>\n

\n

Sedation<\/p>\n<\/td>\n

\n

Sedation<\/p>\n<\/td>\n<\/tr>\n

\n

Body temperature<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n<\/tr>\n

\n

Bodyweight<\/p>\n<\/td>\n

\n

Decreased after one week<\/p>\n<\/td>\n

\n

Decreased after 42 days<\/p>\n<\/td>\n

\n

Normal<\/p>\n<\/td>\n

\n

Reduced by 11%<\/p>\n<\/td>\n<\/tr>\n

\n

Grooming<\/p>\n<\/td>\n

\n

Absent<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n

\n

Present<\/p>\n<\/td>\n<\/tr>\n

\n

Death<\/p>\n<\/td>\n

\n

3 animals were found dead during the testing period<\/p>\n<\/td>\n

\n

No death after 60 days<\/p>\n<\/td>\n

\n

No death<\/p>\n<\/td>\n

\n

No death<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 2: Mean food and water intake of animals. Food (a and b) and water (g and h) intake of animals treated with 1000 mg\/kg up to 28 days. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Histopathology revealed safety of MBZM-N-IBT (50 mg\/kg) <\/strong><\/p>\n\n\n\n

The rat liver tissues (Figures 3a, 4a, and 5a) from the control group showed normal hepatic morphology, but MBZM-N-IBT (1000 mg\/kg for 28 days) treated rat showed activated kupffer cells, infiltration of lymphocytes at the portal\/central vein, sinusoidal obstruction, fibrous connective tissue and necrotic plaques (Figure 3b). In the case of MBZM-N-IBT (500mg\/kg for 60 days), hepatocytes were loosely arranged, and vacuolar degeneration of hepatocytes was observed (Figure 4b). Following MBZM-N-IBT (50 mg\/kg for 270 days) treatment, the liver showed alteration of cytoarchitecture (the arrowhead points to hepatic lobules, where the trabecular structure appears slightly blurred, while the remaining lobules exhibit a distinct blurring). Empty vacuolar spaces were seen in the cytoplasm of some cells. Kupffer cells were additionally detected along the sinusoidal walls (Figure 5b). The kidney tissues (Figures 3c, 4c, and 5c) from the normal control group showed the presence of normal renal corpuscle and kidney tubules. The MBZM-N-IBT (1000 mg\/kg for 28 days) treated rat showed the presence of granular cast, focal proximal tubular epithelial necrosis, and unusual morphological pattern with the degeneration of the renal corpuscles (Figure 3d). Kidney tissues from MBZM-N-IBT (500 mg\/kg for 60 days) treated rats showed cellular cast and Protein and reduction in the renal corpuscles (Figure 4d). The section of the kidney showed a normal appearance (the arrowhead highlights that the collecting tubules are lined with a simple cuboidal epithelium, and there is adjacent interstitial tissue) in case of treatment at 50 mg\/kg\/day for 270 days (Figure 5d). The stomach tissues (Figures 3e, 4e, and 5e) from the normal control group showed the presence of apparent normal mucosa and submucosa and thickened mucosal layer. Stomach tissues from MBZM-N-IBT (1000 mg\/kg for 28 days) treated rats showed minor epithelial ulceration covered by fibrin entangling neutrophils involving the reduction of gastric mucosal thickening (Figure 3f). Whereas 500 mg\/kg for 60 days (Figure 4f) and 50 mg\/kg for 270 days (Figure 5f) treatment showed no alteration in stomach cytoarchitecture of the rats. The lung tissues (Figures 3g, 4g, and 5g) from the normal control group showed polygonal alveoli and blood vessels. The presence of thin interalveolar septa and well-inflated alveoli indicate normal lung architecture. The MBZM-N-IBT (1000 mg\/kg for 28 days) treated rat showed collapsed alveoli, suggesting severe alveolar damage (Figure 3h).<\/p>\n\n\n\n

In contrast,\nrats treated with a lesser dose (500mg\/kg for 60 days) showed slight\ninteralveolar septa thickening. Whereas this suggests a relatively low level of\ntoxicity, isolated focal regions with inflammatory cell infiltration and the\nappearance of clogged, thickened blood vessels indicate adverse effects on the\nlungs with continued treatment (Figure\n4h). Following MBZM-N-IBT (50mg\/kg for 270 days) treatment, normal\nintrapulmonary bronchioles could be seen (Figure 5h). The cardiac tissues (Figures 3i, 4i, and 5i) from the normal\ncontrol group showed no necrosis throughout the musculature, no foci of\ncellular infiltration, normal nuclei, and normal cytoplasm. However, treated rats with MBZM-N-IBT\n(1000 mg\/kg for 28 days) displayed distinct foci of cellular infiltration,\nhaving large nuclei and specific areas that showed general cellular\nultrastructure disruption (Figure 3j). The rat treated with MBZM-N-IBT\n(500mg\/kg for 60 days) displayed loss of nuclei, indicating dead cells and\ninternalization of large nuclei indicative of the myocyte regeneration process\n(Figure 4j). The histological\nsection of the myocardium (Figure 5j) showed a normal appearance (arrowhead\nindicates muscle fiber and blood vessel) in the case of MBZM-N-IBT (50mg\/kg for\n270 days). The brain tissues (Figures 3k, 4k, and 5k) from the normal control\ngroup showed the cerebral cortex with regular ependyma of the ventricle,\npyramidal cells, and perivascular space. The MBZM-N-IBT (1000mg\/kg for 28 days)\ntreated rat showed blood vessels dilated with increased perivascular spaces and\nintervening edema in the form of vacuoles in the brain substance and ischaemic\nnecrosis (Figure 3l). Brain tissues from MBZM-N-IBT (500 mg\/kg for 60 days)\ntreated rats showed pyknotic nuclei and increased perivascular space with\nhemorrhage and vacuoles in the brain (Figure 4l). Meanwhile, the brain tissue (Figure\n5l) showed a normal appearance (arrowhead indicates different sizes and shapes\nof nerve cells and nerve fibers) in the case of MBZM-N-IBT (50 mg\/kg for 270\ndays). This suggests that\nthe dose of 50 mg\/kg\/day is relatively safe for oral administration. <\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 3: Histopathology of rat tissues after the administration of MBZM-N-IBT (1000 mg\/kg) for  28 days.<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 4: Histopathology of rat tissues after the administration of MBZM-N-IBT (500 mg\/kg)  for 60 days. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 5: Histopathology of rat tissues after the administration of MBZM-N-IBT (50 mg\/kg) for 270 days. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Bioanalytical Method Development and Pharmacokinetics of MBZM-N-IBT <\/strong><\/p>\n\n\n\n

A novel RP-HPLC (Reverse Phase High-Performance Liquid\nChromatography) method was established and validated in compliance with the ICH\n(International Council for Harmonization) guidelines, specifically ICH Q2 (R1).\nAll the parameters were validated, and the results are shown in Table 3. After\nthis, the method was verified according to USFDA guidelines for bio-analytical\nmethod validation. Specificity was confirmed by the absence of peaks at the\nretention time of MBZM-N-IBT from the plasma. The calibration curve was\ngenerated by adding a known quantity of MBZM-N-IBT to 500 \u00b5L of rat plasma,\nresulting in concentrations of 2, 4, 6, 8, and 10 \u00b5g\/mL. MBZM-N-IBT in rat plasma\nwas quantified by measuring the analyte’s response against the calibration\ncurve. The concentration of MBZM-N-IBT in six replicates of plasma spiked with\n4, 6, and 8 \u00b5g\/mL of MBZM-N-IBT was evaluated using HPLC in a single day and\nrepeated on three separate days to evaluate accuracy and precision. In\naccordance with FDA guidelines, the acceptance criteria for accuracy were set\nat 85-115%, and the coefficient of variation values (CV) was required to be \u02c2\n15%, except at the Lower Limit of Quantification (LLOQ), where accuracy was\nacceptable within the range of 80-120%, and the CV \u2264 20% (Table 4). The\npharmacokinetic plasma profiles of MBZM-N-IBT after 50 mg\/kg oral\nadministration are depicted in Figure 6, and pharmacokinetic parameters are\nsummarized in Table 5. The estimated maximum plasma concentration (Cmax) was\n1.079\u00b10.069 \u03bcg\/mL, and the duration to achieve the maximum plasma concentration\n(Tmax) was 2h. This shows that absorption of MBZM-N-IBT is not slow and is\nwithin the normal gastric emptying time of 2-4 hours. The\napparent elimination half-life (t1\/2) was calculated as 8.120\u00b10.069h, which\nindicates that more than 40h (five half-lives) may be required for complete\nclearance of MBZM-N-IBT from systemic circulation.<\/p>\n\n\n\n

Table 3: Validation parameter for analysis of MBZM-N-IBT from bulk<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Linearity<\/strong><\/p>\n<\/td>\n

\n

r2<\/sup>=0.999<\/strong><\/p>\n<\/td>\n

\n

0.5-100 \u00b5g\/mL<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Accuracy (%\u00b1SD)<\/p>\n<\/td>\n

\n

 <\/p>\n

100.16 \u00b1 0.557<\/p>\n<\/td>\n<\/tr>\n

\n

Precision<\/p>\n

(RSD %)<\/p>\n<\/td>\n

\n

Intra-day<\/p>\n<\/td>\n

\n

Inter-day<\/p>\n<\/td>\n<\/tr>\n

\n

Day 1<\/p>\n<\/td>\n

\n

Day 2<\/p>\n<\/td>\n

\n

Day 3<\/p>\n<\/td>\n<\/tr>\n

\n

0.722<\/p>\n<\/td>\n

\n

0.55<\/p>\n<\/td>\n

\n

0.57<\/p>\n<\/td>\n

\n

1.31<\/p>\n<\/td>\n<\/tr>\n

\n

LOQ<\/p>\n<\/td>\n

\n

0.150  \u00b5g\/mL<\/p>\n<\/td>\n<\/tr>\n

\n

LOD<\/p>\n<\/td>\n

\n

0.050 \u00b5g\/mL<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 4: Validation of analytical method for analysis of MBZM-N-IBT from plasma.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Linearity<\/p>\n<\/td>\n

\n

r2<\/sup>=0.999<\/p>\n<\/td>\n

\n

2-10 \u00b5g\/ml<\/p>\n<\/td>\n<\/tr>\n

\n

Accuracy (%\u00b1SD)<\/p>\n<\/td>\n

\n

LQC<\/p>\n<\/td>\n

\n

96.7\u00b10.094<\/p>\n<\/td>\n<\/tr>\n

\n

MQC<\/p>\n<\/td>\n

\n

97.5\u00b10.113<\/p>\n<\/td>\n<\/tr>\n

\n

HQC<\/p>\n<\/td>\n

\n

93.9\u00b10.244<\/p>\n<\/td>\n<\/tr>\n

\n

Precision
(RSD %)<\/p>\n<\/td>\n

\n

Intra-day<\/p>\n<\/td>\n

\n

Inter-day<\/p>\n<\/td>\n<\/tr>\n

\n

Day 1<\/p>\n<\/td>\n

\n

Day 2<\/p>\n<\/td>\n

\n

Day 3<\/p>\n<\/td>\n<\/tr>\n

\n

LQC<\/p>\n<\/td>\n

\n

2.55<\/p>\n<\/td>\n

\n

3.36<\/p>\n<\/td>\n

\n

2.85<\/p>\n<\/td>\n

\n

3.31<\/p>\n<\/td>\n<\/tr>\n

\n

MQC<\/p>\n<\/td>\n

\n

1.93<\/p>\n<\/td>\n

\n

3.11<\/p>\n<\/td>\n

\n

3.80<\/p>\n<\/td>\n

\n

2.27<\/p>\n<\/td>\n<\/tr>\n

\n

HQC<\/p>\n<\/td>\n

\n

3.24<\/p>\n<\/td>\n

\n

1.92<\/p>\n<\/td>\n

\n

3.29<\/p>\n<\/td>\n

\n

2.89<\/p>\n<\/td>\n<\/tr>\n

\n

LLOQ<\/p>\n<\/td>\n

\n

2 \u00b5g\/ml<\/p>\n<\/td>\n<\/tr>\n

\n

LOD<\/p>\n<\/td>\n

\n

0.4 \u00b5g\/ml<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 5: Pharmacokinetic parameters of MBZM-N-IBT<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Parameter<\/strong><\/p>\n<\/td>\n

\n

Result<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

AUC (\u00b5g\/mL h)<\/p>\n<\/td>\n

\n

25.604\u00b10.123<\/p>\n<\/td>\n<\/tr>\n

\n

AUMC(\u00b5g\/mL h2<\/sup>)<\/p>\n<\/td>\n

\n

298.346\u00b10.412<\/p>\n<\/td>\n<\/tr>\n

\n

MRT (h)<\/p>\n<\/td>\n

\n

11.652\u00b10.040<\/p>\n<\/td>\n<\/tr>\n

\n

Cmax<\/sub> (\u00b5g\/mL)<\/p>\n<\/td>\n

\n

1.709\u00b10.069<\/p>\n<\/td>\n<\/tr>\n

\n

Tmax<\/sub> (h)<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n<\/tr>\n

\n

Kel<\/sub> (h2<\/sup>)<\/p>\n<\/td>\n

\n

0.085\u00b10.031<\/p>\n<\/td>\n<\/tr>\n

\n

t1\/2<\/sub><\/p>\n<\/td>\n

\n

8.120\u00b10.069<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*mean\u00b1SD (n=6)<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 6: AUC of MBZM-N-IBT in rat plasma.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Plasma protein binding of MBZM-N-IBT <\/strong><\/p>\n\n\n\n

The HPLC method estimated Plasma\nprotein binding using a human-HSA chiral column. The capacity factor measures the\nretention of drugs in the column. Accordingly, the capacity factor of\ndrugs while using a human-HSA chiral column represents its affinity towards\nhuman serum albumin. Since\nthis is the major plasma protein, it\u2019s binding correlates well with drugs’ in vivo<\/em> plasma protein binding. To\ncorroborate this, various drugs with reported plasma protein binding in the\nrange of 1-99% were considered. Their capacity factors were determined (Table 6).\nA significant correlation (r2<\/sup>=0.994) was found between the capacity\nfactor and the known PPB% of these drugs. Accordingly, this showed a linear\nregression (Y= 0.008X+0.144, Figure 7). The modified capacity factor\n(K\u00b4\/(K\u00b4+1)) of MBZM-N-IBT was determined to be 0.94. Accordingly, its PPB% was\nfound to be 99.5 (Table 6). This suggests that MBZM-N-IBT has a high plasma\nprotein binding capacity.<\/p>\n\n\n\n

Table 6: Plasma protein binding % of different drugs<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Drug<\/strong><\/p>\n<\/td>\n

\n

Reported PPB %<\/strong><\/p>\n<\/td>\n

\n

Rt<\/strong><\/p>\n<\/td>\n

\n

K\u00b4<\/strong><\/p>\n<\/td>\n

\n

K\u00b4\/(K\u00b4+1)<\/strong><\/p>\n<\/td>\n

\n

Measured PPB %<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Metformin27<\/sup><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

3.161<\/p>\n<\/td>\n

\n

0.161<\/p>\n<\/td>\n

\n

0.138<\/p>\n<\/td>\n

\n

0.00<\/p>\n<\/td>\n<\/tr>\n

\n

Metronidazole28<\/sup><\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

3.546<\/p>\n<\/td>\n

\n

0.244<\/p>\n<\/td>\n

\n

0.196<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

Ranitidine29<\/sup><\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

4.032<\/p>\n<\/td>\n

\n

0.482<\/p>\n<\/td>\n

\n

0.325<\/p>\n<\/td>\n

\n

22.625<\/p>\n<\/td>\n<\/tr>\n

\n

Paracetamol30<\/sup><\/p>\n<\/td>\n

\n

25<\/p>\n<\/td>\n

\n

4.494<\/p>\n<\/td>\n

\n

0.571<\/p>\n<\/td>\n

\n

0.363<\/p>\n<\/td>\n

\n

27.375<\/p>\n<\/td>\n<\/tr>\n

\n

Phenobarbital31<\/sup><\/p>\n<\/td>\n

\n

45<\/p>\n<\/td>\n

\n

6.44<\/p>\n<\/td>\n

\n

1.027<\/p>\n<\/td>\n

\n

0.506<\/p>\n<\/td>\n

\n

45.25<\/p>\n<\/td>\n<\/tr>\n

\n

Cefixime32<\/sup><\/p>\n<\/td>\n

\n

67<\/p>\n<\/td>\n

\n

2.798<\/p>\n<\/td>\n

\n

2.436<\/p>\n<\/td>\n

\n

0.708<\/p>\n<\/td>\n

\n

70.05<\/p>\n<\/td>\n<\/tr>\n

\n

Salicylic acid33<\/sup><\/p>\n<\/td>\n

\n

85<\/p>\n<\/td>\n

\n

15.610<\/p>\n<\/td>\n

\n

8.974<\/p>\n<\/td>\n

\n

0.899<\/p>\n<\/td>\n

\n

94.375<\/p>\n<\/td>\n<\/tr>\n

\n

Cetrizine34<\/sup><\/p>\n<\/td>\n

\n

93<\/p>\n<\/td>\n

\n

28.662<\/p>\n<\/td>\n

\n

16.834<\/p>\n<\/td>\n

\n

0.943<\/p>\n<\/td>\n

\n

99.875<\/p>\n<\/td>\n<\/tr>\n

\n

Omeprazole35<\/sup><\/p>\n<\/td>\n

\n

95<\/p>\n<\/td>\n

\n

40.638<\/p>\n<\/td>\n

\n

24.414<\/p>\n<\/td>\n

\n

0.96<\/p>\n<\/td>\n

\n

102<\/p>\n<\/td>\n<\/tr>\n

\n

Losartan36<\/sup><\/p>\n<\/td>\n

\n

99<\/p>\n<\/td>\n

\n

52.971<\/p>\n<\/td>\n

\n

32.985<\/p>\n<\/td>\n

\n

0.970<\/p>\n<\/td>\n

\n

103.25<\/p>\n<\/td>\n<\/tr>\n

\n

MBZM-N-IBT<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

51.962<\/p>\n<\/td>\n

\n

17.248<\/p>\n<\/td>\n

\n

0.94<\/p>\n<\/td>\n

\n

99.5<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 7: Calibration curve for Plasma protein binding <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Metabolites of MBZM-N-IBT showed no toxicity.<\/strong><\/p>\n\n\n\n

No\nmetabolite was found for metabolism in the autoxidation and dissociation\nsimulator, indicating the molecule’s stability. A simulator for hydrolysis under neutral conditions\npredicted 5 metabolites. Under acidic and basic conditions, the hydrolysis simulator predicted\n12 and 13 metabolites, respectively. In the in vivo<\/em> rat simulator, 13\nmetabolites were predicted. After combining the results of all these\nsimulators, 22 unique metabolites were predicted for MBZM-N-IBT (Table 7).\nWhile 21 metabolites were expected to be OECD class five chemicals with very\nhigh LD50, one metabolite was categorized as OECD class 4 with a potential LD50\nof 681mg\/kg. This indicates that the metabolites of MBZM-N-IBT are not likely\nto be toxic.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 7: Predicted Metabolites of MBZM-N-IBT.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

MBZM-N-IBT is an important antiviral lead. It has a broad spectrum of\nantiviral action and the potential to interfere in multiple ways against CHIKV\nand HSV replication6,11<\/sup>.\nHowever, it has been demonstrated to be effective in vivo at a dosage of 15\nmg\/kg against CHIKV infection in mice; further preclinical validation is\nnecessary to justify its suitability as an antiviral candidate37<\/sup>. Hence, assessing its pharmacokinetics and toxicology is\nessential to encourage further in vivo<\/em>\nvalidation. <\/p>\n\n\n\n

An oral acute toxicity study, as per OECD-423 guidelines,\nrevealed that rats had no acute toxicity during the monitoring period (14 days)\n(Table 1). Accordingly, the oral LD50<\/sub> of the\nMBZM-N-IBT was calculated to be greater than 5000 mg\/kg as per the OECD-423\nguidelines. This suggested the acute oral safety of MBZM-N-IBT. In the chronic toxicity studies, MBZM-N-IBT administration\nat a high dose for 28 days (1000 mg\/kg\/day) led to a remarkable reduction in food intake and water consumption.\nAlthough this was normal in the first three weeks in animals treated with a\nmoderate dose (500 mg\/kg\/day), in the later stages, there was a reduction in\nfood intake and water consumption (Figure 2). At a lower dose (50 mg\/kg\/day),\nrats showed no significant change in food intake and water consumption for 270\ndays. Also, there were no signs of toxicity and behavioural changes (Table 2).\nHowever, on prolonged use, some reversible sedation was noted. Besides, there\nwere some changes in stool and body weight (Table 2). In congruence with these,\nsignificant alterations were observed in the pathological\nstudy of the excised liver, lungs, kidney, stomach,\nheart, and brain (Figures 3 & 4). At the end of the 270-day study, some\nnecrosis of excised cardiomyocytes at the sub-endocardium region in cardiac\ntissue was observed. In excised brain tissue, vascular congestion, and cellular\ninfiltration were also marked (Figure 5).<\/p>\n\n\n\n

Nevertheless,\nthe pathological study of the excised liver, kidney, stomach, and lung tissue\nshowed a normal appearance (Figure 5). The sedation on prolonged use suggests\nthe ability to reach the brain. Nevertheless, till 120 days and beyond, it\nshowed no effect on motor coordination, locomotion, and\nanxiety at a dose of 50 mg\/kg. Since the antiviral\napplication usually requires short-term drug administration, this study hints\nat the safety of this dose for further investigation.<\/p>\n\n\n\n

The\nanalytical and bio-analytical method validation parameters are shown in Tables\n3 and 4. Following an oral dose of 50 mg\/kg to rats, the AUC of MBZM-N-IBT was\ndetermined (Figure 6). It revealed that 2h is taken to achieve the highest\nsystemic concentration. This shows that it is absorbed within 2h of oral\nadministration. Considering\nthe fact that the normal gastric emptying time is between 2-4h, the absorption\nrate of MBZM-N-IBT may be adequate.<\/p>\n\n\n\n

Further,\nits elimination half-life was found to be 8 h (Table 5). Drugs with a half-life\nof 12-48 h are generally considered suitable for once-daily oral dosing38<\/sup>. Although the half-life of\nMBZM-N-IBT is less than this, it can remain in circulation for 5 half-lives or\n40h. Hence, its suitability for once-daily oral dosing cannot be ruled\nout without further validation. Moreover, it has been demonstrated to be\neffective against CHIKV infection in mice with a once-daily oral dosing of 15\nmg\/kg37<\/sup>.<\/p>\n\n\n\n

The\nprotein binding was estimated using the HPLC method by measuring its affinity for\nhuman serum albumin attached to the stationary phase. To develop this method,\nthe modified capacity of different drugs that represent their affinity for\nhuman serum albumin was correlated to their known PPB%. A high correlation (r2<\/sup>\n= 0.994) suggested the suitability of the regression equation that was used for\nthe calculation of PPB% (Figure 7). As shown in Table 6, the calculated PPB%\nwas very close to that of the reported values for most of the drugs. Hence,\n99.5% PPB was estimated for MBZM-N-IBT. This high PPB is in agreement with the\nrelatively high elimination half-life (8h) and may support the longer duration\nof action.<\/p>\n\n\n\n

Further,\nthe metabolism of MBZM-N-IBT was predicted through in silico<\/em> analysis using the OECD QSAR Toolbox.<\/em> The reliability of the OECD QSAR toolbox\nhas been demonstrated for the prediction of the acute toxicity of organic\nchemicals39<\/sup>. It is supported by the Organisation for Economic\nCo-operation and Development (OECD) as well as the European Chemicals Agency (ECHA). Using these tools, 22 unique metabolites\n(Table 7) were predicted. While\n21 of these metabolites were non-toxic (OECD-class 5), one was predicted to\nhave an LD50 of 681mg\/kg (Table 7). Thus, like MBZM-N-IBT, the metabolites are\nalso not likely to be toxic. Since these metabolites were mainly predicted in\nthe rat simulator model, this may also support the safety in rats demonstrated\nin the chronic toxicity studies. However, further in vivo<\/em> metabolite\nanalysis is necessary to validate this. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The no-observed-adverse-effect level (NOAEL) of MBZM-N-IBT can be\ntaken into consideration to be 50 mg\/kg under the specified circumstances based\non the single-dose oral acute toxicity study and the lack of substantial\ntoxicity or mortality in the sub-chronic toxicity study. While the in vivo<\/em> pharmacokinetics suggests\nadequate rates of absorption, elimination half-life (8h), and high PPB suggest\na potentially longer duration of action in\nvivo<\/em>. The predicted metabolites are not expected to be toxic and support\nthe in vivo<\/em> safety of MBZM-N-IBT\nobserved in toxicity studies. However, it is necessary to closely evaluate its\neffect on renal and hepatic function for long-term therapy. Although at this\ndose, it does not affect motor coordination, locomotion, and anxiety, some\nsedation is observed. Hence, it is necessary to closely monitor its effect on\nthe brain. Further investigation into mutagenicity and teratogenicity is also\nrequired. Thus, the present\ninvestigation can encourage additional studies to increase the scope for\ntranslational application of MBZM-N-IBT.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

This work was\nsupported by the Department of Biotechnology (DBT), Ministry of Science and\nTechnology, Government of India.  <\/p>\n\n\n\n

Conflict of interests<\/strong><\/p>\n\n\n\n

The authors declare that\nthey have no known competing financial interests or personal relationships that\ncould have appeared to influence the work reported in this paper.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

This work was supported by\nthe Grant BT\/PR15750\/MED\/29\/1015\/2016 from the Department of Biotechnology\n(DBT), Ministry of Science and Technology, Government of India. <\/p>\n\n\n\n

Ethical Approval<\/strong><\/p>\n\n\n\n

The experimental procedure was approved by the Institutional Animal Ethics Committee (IAEC) of School of\nPharmaceutical Sciences, Siksha O Anusandhan (Deemed to be University),\nBhubaneswar, India (Reg. no. 1171\/PO\/Re\/S\/08\/ CPCSEA).<\/p>\n\n\n\n

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