Development, characterization and pharmacological evaluation of amino acid prodrugs of (+)-Ibuprofen

The current research work investigate the neuronal pathway associated with NSAIDs that lead to the reduction in the initiation and progression of neurodegenerative diseases such as Alzheimer’s disease, Parkinsonism etc. This research was developed amino acid prodrugs of the active enantiomer of ibuprofen, (+)-IBN and checks the pharmacological effects, neuroprotective effect, anti in lammatory effect and anti-ulcerogenic effect. The treatment using (+)-IBN reduced the action of micoglia and the release of cytokine especially TNFα that are mainly involved in the neurodegenerative process. (+)IBN reduced the deposition of soluble beta amyloid plaque by inhibiting the amyloid precursor protein, beta-secretase 1 and also enhancing the degradation process of beta amyloid via induction of insulin degrading enzyme. (+)-IBN also showed the property that the reduction in the TAU misfolding process. Therefore, the synthesized (+)-IBN amino acid prodrugs treatment effectively produce neuroprotective action, both restoringmemory realed risk factors and reversing multiple brain neuropathological hallmarks. The current research study developed the three amino acid prodrugs of (+)-ibuprofen by conjugating with L-phenyl alanine, L-tryptophan and L-tyrosine also the physico-chemical characterization and spectral characterization was done. This study modify the carboxylic acid functional group present in the NSAIDs that lead to the formation of prodrugswith enhanced anti-in lammatory activity, reduced side effects and protective effect against neurodegenerative processes.


INTRODUCTION
The general pharmacological activities of Nonsteroidal anti-in lammatory drugs (NSAIDs) are analgesic activity, antipyretic activity and anti in lammatory activity. The general mechanism of NSAIDs is explained through the inhibition of prostaglandins(PGs) by the action of two cyclooxygenase enzymes. PGs having signi icant role in various cellular activities like gastrointestinal protection, in lammation, angiogenesis, hemostasis etc. (Gunaydin and Bilge, 2018). Many studies have been conducted for investigating the different pharmacological and therapeutical activities of NSAIDs. Studies revealed the signi icance of NSAIDs in various activities and treatment strategies like       anticancer activity, anti-Alzheimer's activity, antiparkinsonian activity etc. (Osafo and Agyare, 2017;Rebecca and Wong, 2019;Ettcheto et al., 2017;Ajmone-Cat et al., 2010).
The current research aims to develop the novel amino acid prodrugs of NSAID, (+)-IBN and investigate the effect of NSAIDs and their conjugates in different areas. The use of prodrugs based approach modify the carboxylic acid functional group of NSAIDs that produced the compounds with better physical-chemical properties, pharmacokineticpharmacodynamic properties, pharmacological activities and reduced toxicity pro ile (Rasheed et al., 2011;P et al., 2018).
Literature survey showed that the long term use of NSAIDs reduce the risk and progression of neuro-degeneration by the signi icant effect on the microglial activity, reduction in beta amyloid production, facilitate the Aβ degradation, reduction in the process of TAU protein hyperphosphorylation and reduced the problems like spatial learning and memory impairment (Khansari and Halliwell, 2019;Španić et al., 2019). So this study synthesize the three amino acid prodrugs and expect to give improved properties (Rasheed and Kumar, 2008).

Reagent and chemicals
The amino acids were obtained from Hi-media chemicals, India, (+)-IBN was obtained from Alkem laboratories, Mumbai and all other reagents are analytical grade. The melting points were monitored by the Melting point apparatus and the elemental analysis was done in CDRI, Lucknow. The infra red spectra were recorded by IR spectrometer, Al Shifa College of pharmacy. 1 H NMR, 13 C NMR and Mass spectra were recorded in IICB, Kolkata. The homogenate was prepared by using the homogeniser, Remi instruments division. The histopathological studies were carried out in Department of Pathology, KIMS Al-shifa hospital Kerala. The cell line studies of neurodegenerative disease were carried out in Biogenix, Trivandrum.

Physical-chemical characterization methods
The progression of the reaction and the purity of the compounds con irmed by thin layer chromatography on pre-coated silica-G plates and the detection method is UV chamber visualization (Rao and Shil, 2019;Kemp, 1991). The melting point of the drug and prodrugs were done for con irming the product formation and it was done by using melting point apparatus (John and Young, 2013) . Solubility of the drug and amide prodrugs were done in different solvents to identify the nature of the compounds (Qiu et al., 2009). The quanti ication of the elements present in the synthesized compounds and it was compared with that of the calculate values are signi icant method to con irm the successful synthesis (Margui and Grieken, 2014). The partition coef icient of the podrugs were done by shake falsk method explained by (Paschke et al., 2004). Protein binding of the drugs and prodrugs were to con irm that the drug is available for the pharmacological activity and it was done by equilibrium dialysis method explained by (Pinger et al., 2017;Barton et al., 2007). Spectral characterization was very much important to con irm the desired structures and done by IR, 1 HNMR, 13 CNMR and Mass spectroscopy (Silverstein and Webster, 1998;Jurgen, 2004).

In silico Studies
ADME Optimization is signi icant in the drug development process that was done by QikProp according to the Schrödinger [Internet], USA (Schrödinger, 2015).

Biodistribution and pharmacokinetic studies
The best distributed prodrug in the brain can be found out by bio-distribution study and that can be monitored by the parameter brain targeting ef iciency. The procedure was according to the (Zhang et al., 2012).

Pharmacological evaluation
The activties tested were anti-in lammatory activity,ulcerogenecity and protective effect in brain. In brain, (+)-IBN and its synthesized prodrugs were evaluated for behavioral parameters, antioxidant parameter, histopathology of the brain cortex and the results were compared with that of the parent drug. Neurotoxicity was induced by aluminium induced model by the oral administration of aluminium chloride [100mg/kg/day] for three months (Shati et al., 2011). The animals were grouped in to six groups and each group contained six animals. The irst group acts as control that receives normal saline. The second group received aluminium chloride only. The third, fourth, ifth, sixth group received (+)-IBN [1. Behavioral studies

Open f ield tests
The open ield exploration study was carry out according to the procedure explained by (Taïr et al., 2016)

Water maze task
This test access the spatial learning and memory conducted as per the procedure followed by (Vorhees and Williams, 2006;Nunez, 2008).

Biochemical estimations
Antioxidant parameters were estimated by testing the activity of the enzyme catalase and superoxide dismutase (SOD) (Pham-Huy et al., 2008). SOD activity was done by the procedure explained by (Weydert and Cullen, 2010) and the catalase activity was done as per the procedure (Goth, 1991;Hadwan and Abed, 2016).

Histopathological Studies
The cortex of the brain was collected and histopathologically evaluated by using haematoxylin-eosin dye in Department of pathology, KIMS-Al shifa hospital,kerala and The sections were examined microscopically for histopathological changes observed and compare with that of parent NSAID (Nobakht et al., 2011).

Anti -in lamamtory activity and ulcerogenecity study
Anti in lammatory activity was performed by carrageenan induced paw edema method explained by (Khedir et al., 2016). The main side effect of the NSAIDs is the production of ulcers that were studied and monitored as per the procedure by (Rasheed et al., 2016).

Statistical analysis
Statistical signi icance was done by ANOVA and the values were expressed as mean ± SD.

Physical and chemical characterization methods
The nature of the prodrugs was evaluated by various methods. Different methods adopted and their results were given in the Table 1. The data in the table revealed that the formation of pure amide prodrugs. The melting point determination and thin layer chromatography results showed the formation of the products. The solubility studies proved the organic solubility of the prodrugs that showed its Figure 13: Comparative ulcerogenecity of (+)-IBN and its prodrugs lipophilic nature. Also the Log P value calculated from the partition coef icient method revealed the enhancement of lipophilic pro ile of the amide prodrugs. The lower protein binding values indicate the availability of the drug in various biological luids. Elemental analysis data provided the comparable values with the theoretical values spectral data con irm the structure of the compounds with amide and ester linkages.

Insilico studies
The ADME optimization is very much signi icant and that was done by insilico tool. Data was given in Table ??.
In Table ?? shows, a Predicted octanol/water partition co-ef icient log p (acceptable range: −2.0 to 6.5). b Predicted Caco-2 cell permeability in nm/s (acceptable range, <25 is poor and >500 is great). c Predicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5). d Predicted apparent MDCK cell permeability for the blood-brain barrier, (acceptable range, < 25 is poor and >500 is great) , e Percentage of human oral absorption (< 25% is poor and >80% is high).

Biodistribution and Pharmcokine tics
The drug with more distribution in the brain and Brain targeting ef iciency (BTE) can be found out from the biodistribution study and the data was presented in Table 3 and graphically represented in Figures 2 and 3. (+)-IBN 2 showed higher BTE value and brain distribution shown in Figure 3. So Pharmacokinetic studies were carried out using (+)-IBN and (+)-IBN 2 .The concentration-time pro ile of plasma and brain and BTE was given in Table 4 graphically represented in Figures 4 and 5 respectively. The targeting ef iciency parameters are presented in Table 5. The study revealed that the concentration of the prodrug after 10 minutes of admin-istration is very high in brain compared to that of (+)-IBN. The brain targeting ef iciency of the prodrugs were high compared to that of (+)-IBN. Also, the RE,CE and DTI showed the remarkable high distribution of (+)-IBN 2 compared to that of (+)-IBN.

Pharmacological Evaluation
Behavioral parameters were monitored by Open ield test [ Figure 6], Marble burying test [ Figure 7], and Water maze test [ Figure 8]. In open ield test, head dipping, rearing and line crossing were considered for monitoring the cognitive activity. Behavioral data obtained from the marble burying test is a parameter to test the locomotion. The behavioral studies proved that the enhancement in the locomotion, memory, spatial learning etc shown by the prodrugs compared to that of the parent NSAID that indicates that the potential activity of (+)-IBN is nil due to the inability to cross BBB.

Biochemical Estimation
The evaluation of antioxidant parameters indicated the protective effect of the synthesized amide prodrugs against neurodegeneration.SOD and Catalase activity were graphically represented in Figure 9.

Histopathological analysis
The histopathology study of the brain cortex was taken and concluded that the prodrug treated group, all the three different aminoacid prodrugs shown the normal architecture of the cells without any spongiform cells. The study proved the protective nature of the NSAIDs shown in Figure 10.

Invitro neuroprotective effect determination by MTT assay
From the cell line studies, the effects of the samples (+)-IBN-2 on H 2 O 2 induced cell death was examined in SH-SY5Y cells. As illustrated in the Figure 11 at a concentration of 25 µg.mL −1 all two samples are having a neuroprotective activity by measuring the percentage viability.

Anti In lammatory Activity
The percentage anti in lammatory activity of (+)-IBN and its prodrugs were determined and are given in Table 6. A graphical representation was shown in Figure 12. From the study concluded that the synergistic activity pro ile of the synthesized amide prodrugs with the signi icance of < 0.05. After the six hours observations the amide prodrugs showed about 70 percentage of anti in lammatory activity that is very high compared to that of the parent (+)-IBN.

CONCLUSIONS
This research study proved that the new amide prodrugs showed better physico chemical properties, transport properties, bio-distribution and pharmacological activities. On the basis of the results, it can be concluded that prodrug approach could successfully attain the goal of improving the transport properties through the BBB and protective effect against the degeneration in brain. The prodrug approach on the NSAIDs provides the improvement in the anti in lammatory activity, reduction in the gastric toxicity and behavioral test, antioxidant parameters revealed the protective effect in the brain. The study revealed that the new synthesized prodrugs showed better lipophilicity, brain targeting ef iciency, drug targeting index, relative ef iciency and concentration ef iciency, anti in lammatory activity and anti ulcerogenecity than the parent drug. On the basis of the results, concluded that the prodrug based drug design lead to the formation of the products with the enhancement of the properties.

Funding Support
None.

Con lict of Interest
None.