CYP7B1 as a Biomarker for Prostate Cancer Risk and Progression: Metabolic and Oncogenic Signatures (Diagnostic Immunohistochemistry Analysis by Tissue Microarray in Prostate Cancer Patients-Diamond Study).

We aimed to analyze the association between CYP7B1 and prostate cancer, along with its association with proteins involved in cancer and metabolic processes. A retrospective analysis was performed on 390 patients with prostate cancer (PC) or benign prostatic hyperplasia (BPH). We investigated the interactions between CYP7B1 expression and proteins associated with PC and metabolic processes, followed by an analysis of the risk of biochemical recurrence based on CYP7B1 expression. Of the 139 patients with elevated CYP7B1 expression, 92.8% had prostate cancer. Overall, no increased risk of biochemical recurrence was associated with CYP7B1 expression. However, in a non-diabetic subgroup analysis, higher CYP7B1 expression indicated a higher risk of biochemical recurrence, with an HR of 1.78 (CI: 1.0-3.2, p = 0.05). PC is associated with elevated CYP7B1 expression. In a subgroup analysis of non-diabetic patients, elevated CYP7B1 expression was associated with an increased risk of biochemical recurrence, suggesting increased cancer aggressiveness.

Phase angle at bioelectric impedance analysis is associated with detrimental sperm quality in idiopathic male infertility: a preliminary clinical study.

Background: In 2020, 38% of adults were affected by obesity, while infertility globally affected 1 in 6 people at some stage of their lives.Body mass index (BMI) provides an easy but occasionally inaccurate estimation of body composition. To achieve a more precise assessment, bioelectric impedance analysis serves as a validated tool that administers electrical energy through surface electrodes. Phase angle as a function of the relationship between tissues resistance and reactance, is a trustworthy predictor of body composition and cell membrane integrity. Objectives: We aim to assess whether there is an association between phase angle and seminal parameters, as well as sperm DNA fragmentation percentage. Design: Semen samples of 520 idiopathic infertile patients were analyzed according to 2021 World Health Organization guidelines and evaluated for sperm DNA fragmentation rate. Each participants underwent bioelectric impedance analysis. Results: Median age was 40 years old, median BMI was 26.3 kg/m2, median phase angle was 6.2°. In the logistic regression analysis adjusted for age and total intracorporeal water, phase angle (continuous) was significantly associated with oligozoospermia (odds ratio [OR]:0.4; p<0.01) and sperm morphology (OR: 0.65; p=0.05) and slightly with sperm DNA fragmentation (OR: 0.98; p=0.07). In subgroup analysis, the logistic regression analysis adjusted for the mentioned parameters showed that a phase angle between 6.2 and 7 (°) (OR: 0.63; p=0.02) and >7 (°) (OR: 0.12; p<0.01) were associated with a reduced risk of oligozoospermia compared to values <6.2 (°). Similarly, a phase angle between 6.2 and 7 (°) (OR: 0.57; p< 0.01 and OR: 0.58; p= 0.01) and PA > 7 (°) (OR: 0.12; p= 0.03 and OR: 0.21; p< 0.01) were associated with a reduced risk of lower sperm concentration and lower total sperm count, respectively, compared to a phase angle < 6.2 (°). Conclusion: Our study suggests a negative association between phase angle and detrimental sperm parameters in male idiopathic infertility.

Reactive intermediates formation and bioactivation pathways of spebrutinib revealed by LC-MS/MS: In vitro and in silico metabolic study.

Spebrutinib is a new Bruton tyrosine kinase inhibitor developed by Avila Therapeutics and Celgene. Spebrutinib (SPB) is currently in phase Ib clinical trials for the treatment of lymphoma in the United States. Preliminary in-silico studies were first performed to predict susceptible sites of metabolism, reactivity pathways and structural alerts for toxicities by StarDrop WhichP450™ module, Xenosite web predictor tool and DEREK software; respectively. SPB metabolites and adducts were characterized in vitro from rat liver microsomes (RLM) using LC-MS/MS. Formation of reactive intermediates was investigated using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles for the unstable and reactive iminium, iminoquinone and aldehyde intermediates, respectively, with the aim to produce stable adducts that can be detected and characterized using mass spectrometry. Fourteen phase I metabolites, four cyanide adducts, six GSH adducts and three methoxylamine adducts of SPB were identified and characterized. The proposed metabolic pathways involved in generation of phase I metabolites of SPB are oxidation, hydroxylation, o-dealkylation, epoxidation, defluorination and reduction. Several in vitro reactive intermediates were identified and characterized, the formation of which can aid in explaining the adverse drug reactions of SPB. Several iminium, 2-iminopyrimidin-5(2H)-one and aldehyde intermediates of SPB were revealed. Acrylamide is identified as a structural alert for toxicity by DEREK report and was found to be involved in the formation of several glycidamide and aldehyde reactive intermediates.

Mechanism of Action of Natural Compounds in Peripheral Multiorgan Dysfunction and Hippocampal Neuroinflammation Induced by Sepsis.

Bacterial sepsis induces the production of excessive pro-inflammatory cytokines and oxidative stress, resulting in tissue injury and hyperinflammation. Patients recovering from sepsis have increased rates of central nervous system (CNS) morbidities, which are linked to long-term cognitive impairment, such as neurodegenerative pathologies. This paper focuses on the tissue injury and hyperinflammation observed in the acute phase of sepsis and on the development of long-term neuroinflammation associated with septicemia. Here we evaluate the effects of Coriolus versicolor administration as a novel approach to treat polymicrobial sepsis. Rats underwent cecal ligation and perforation (CLP), and Coriolus versicolor (200 mg/kg in saline) was administered daily by gavage. Survival was monitored, and tissues from vital organs that easily succumb to infection were harvested after 72 h to evaluate the histological changes. Twenty-eight days after CLP, behavioral analyses were performed, and serum and brain (hippocampus) samples were harvested at four weeks from surgery. Coriolus versicolor increased survival and reduced acute tissue injury. Indeed, it reduced the release of pro-inflammatory cytokines in the bloodstream, leading to a reduced chronic inflammation. In the hippocampus, Coriolus versicolor administration restored tight junction expressions, reduce cytokines accumulation and glia activation. It also reduced toll-like receptor 4 (TLR4) and neuronal nitric oxide synthase (nNOS) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome components expression. Coriolus versicolor showed antioxidant activities, restoring glutathione (GSH) levels and catalase and superoxide dismutase (SOD) activities and reducing lipid peroxidation, nitrite and reactive oxygen species (ROS) levels. Importantly, Coriolus versicolor reduced amyloid precursor protein (APP), phosphorylated-Tau (p-Tau), pathologically phosphorylated tau (PHF1), phosphorylated tau (Ser202 and Thr205) (AT8), interferon-induced transmembrane protein 3 (IFITM3) expression, and ß-amyloid accumulation induced by CLP. Indeed, Coriolus versicolor restored synaptic dysfunction and behavioral alterations. This research shows the effects of Coriolus versicolor administration on the long-term development of neuroinflammation and brain dysfunction induced by sepsis. Overall, our results demonstrated that Coriolus versicolor administration was able to counteract the degenerative process triggered by sepsis.

Spectroscopic, molecular docking and dynamic simulation studies of binding between the new anticancer agent olmutinib and human serum albumin.

In this study, the interaction between human serum albumin (HSA), which is the key bio-distributor of exogenous and endogenous compounds in the human bloodstream, and HM61713 (Olmutinib; OMB), which is used as an anticancer drug, is examined by multiple spectroscopic techniques (steady-state fluorescence, UV spectrophotometry, synchronous, and 3 D fluorescence) combined with molecular docking and molecular dynamic simulation investigations. The fluorescence results clearly demonstrated quenching in HSA fluorescence in the existence of OMB indicating the formation of complex and have also shown that the interaction is static. Fluorescence spectroscopy was used to obtain the binding constant values that revealed a strong interaction between the HSA and OMB at 298 K with a binding constant of 7.39x104 M-1 suggesting strong interaction. OMB binds to HSA at site I (IIA). Electrostatic forces and H-bonding were the main binding forces of main bonding between HSA and OMB as revealed by docking and thermodynamic results.Communicated by Ramaswamy H. Sarma.

Biophysical insight into the interaction of levocabastine with human serum albumin: spectroscopy and molecular docking approach.

Interaction of levocabastine with human serum albumin (HSA) is investigated by applying fluorescence spectroscopy, circular dichroism spectroscopy and molecular docking methods. Levocabastine is an important drug in treatment of allergy and currently a target drug for drug repurposing to treat other diseases like vernal keratoconjuctivitis. Fluorescence quenching data revealed that levocabastine bind weakly to protein with binding constant in the order of 103 M-1. Förster resonance energy transfer results indicated the binding distance of 2.28 nm for levocabastine. Synchronous fluorescence result suggest slight blue shift for tryptophan upon levocabastine binding, binding of levocabastine impelled rise in α-helical structure in protein, while there are minimal changes in tertiary structure in protein. Moreover, docking results indicate levocabastine binds to pocket near to the drug site-I in HSA via hydrogen bonding and hydrophobic interactions. Understanding the interaction of levocabastine with HSA is significant for the advancement of therapeutic and diagnostic strategies for optimal treatment results.Communicated by Ramaswamy H. Sarma.

Biophysical Insight into the Interaction of Human Lysozyme with Anticancer Drug Anastrozole: A Multitechnique Approach.

In the present study, we employ fluorescence spectroscopy, dynamic light scattering, and molecular docking methods. Binding of anticancer drug anastrozole with human lysozyme (HL) is studied. Binding of anastrozole to HL is moderate but spontaneous. There is anastrozole persuaded hydrodynamic change in HL, leading to molecular compaction. Binding of anastrozole to HL also decreased in vitro lytic activity of HL. Molecular docking results suggest the electrostatic interactions and van der Waals forces played key role in binding interaction of anastrozole near the catalytic site. Binding interaction of anastrozole to proteins other than major transport proteins in blood can significantly affect pharmacokinetics of this molecule. Hence, rationalizing drug dosage is important. This study also points to unrelated effects that small molecules bring in the body that are considerable and need thorough investigation.

Spectroscopic and molecular docking studies reveal binding characteristics of nazartinib (EGF816) to human serum albumin.

The interactions of novel anti-cancer therapeutic agents with the different plasma and tissue components, specifically serum albumins, have lately gained considerable attention due to the significant influence of such interactions on the pharmacokinetics and/or -dynamics of this important class of therapeutics. Nazartinib (EGF 816; NAZ) is a new anti-cancer candidate proposed as a third-generation epidermal growth factor receptor tyrosine kinase inhibitor that is being developed and clinically tested for the management of non-small cell lung cancer. The current study aimed to characterize the interaction between NAZ and human serum albumin (HSA) using experimental and theoretical approaches. Experimental results of fluorescence quenching of HSA induced by NAZ revealed the development of a statically formed complex between NAZ and HSA. Interpretation of the observed fluorescence data using Stern-Volmer, Lineweaver-Burk and double-log formulae resulted in binding constants for HSA-NAZ complex in the range of (2.34-2.81) × 104 M-1 over the studied temperatures. These computed values were further used to elucidate thermodynamic attributes of the interaction, which showed that NAZ spontaneously binds to HSA with a postulated electrostatic force-driven interaction. This was further verified by theoretical examination of the NAZ docking on the HSA surface that revealed an HSA-NAZ complex where NAZ is bound to HSA Sudlow site I driven by hydrogen bonding in addition to electrostatic forces in the form of pi-H bond. The HSA binding pocket for NAZ was shown to encompass ARG 257, ARG 222, LYS 199 and GLU 292 with a total binding energy of -25.59 kJ mol-1.

Deciphering the enhanced inhibitory, disaggregating and cytoprotective potential of promethazine towards amyloid fibrillation.

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC50) of PRM for HI and HL was estimated to be 114.81±1.21µM and 186.20±1.03µM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC50) was calculated as 21.37±0.89µM and 45.70±0.76µM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases.

Biophysical insight reveals tannic acid as amyloid inducer and conformation transformer from amorphous to amyloid aggregates in Concanavalin A (ConA).

The aggregation phenomenon (amyloid and amorphous) is associated with several pathological complications in human, such as Alzheimer's, Parkinson's, Huntington, Cataract diseases, and Diabetes mellitus type 2. In the present study we are offering evidence and breaking the general belief with regard to the polyphenols action as protein aggregate inhibitors. Herein we confirm that tannic acid (TA) is not only an amyloid inducer, but also it switches one type of conformation, ultimately morphology, into another. We ascertain based on our findings that aggregates are not rigid structures and the stability can be challenged under certain conditions. This study also confirms that unfolded and amorphous aggregates can serve as precursors of amyloids and TA interactions with unordered aggregates (amorphous) bringing orderliness in the conformation via amyloidosis. The shifting of unordered conformation toward orderliness is governed by the modulation in surface hydrophobic patches in Concanavalin A (ConA). Hence, a degree of exposed hydrophobic cluster can be claimed as a strong parameter to detect and distinguish the native, amorphous and both types of amyloids. Turbidity and Rayleigh light scattering measurements followed similar pattern while Thioflavin T and 1-anilino-8-naphthalene sulfonate fluorescence assays of the binding with amorphous and amyloid followed an inverse relation. Electron microscopic studies revealed the morphological variation in the ConA at 65°C as amorphous while the ConA treated with TA followed by heat treatment at 65°C was defined as amyloid in nature. Interestingly for the first time we are reporting the slight agglutination activity by the ConA amyloids.

Investigating the site selective binding of busulfan to human serum albumin: Biophysical and molecular docking approaches.

We have studied the binding of busulfan (BN) to human serum albumin (HSA) at physiological pH 7.4 by using fluorescence, UV-vis and circular dichroism (CD) spectroscopic tools, as well as dynamic light scattering (DLS) measurements and molecular simulation approaches. HSA fluorescence quenching experiments showed that BN reduces the HSA native fluorescence intensity through the static mechanism. In addition, a single binding site on the HSA is occupied by BN with a binding constant at 298K of 1.84×103M-1. The enthalpy change (ΔH) and entropy change (ΔS) of BN-HSA interaction were calculated as -1.40kcalmol-1 and +10.14calmol-1K-1 respectively, which suggest the possible interaction mode as hydrophobic and hydrogen bonding. Moreover, the secondary structure alteration of HSA following its complexation with BN was studied and showed that α-helical content of HSA gets increased on interacting with BN. Ligand binding site to HSA was further investigated by site-specific markers in fluorescence measurements as well molecular modeling approach which indicated that BN bind to the nearby sudlow site II of HSA through hydrophobic as well as hydrogen bonding interaction. The present study will be helpful for understanding the binding mechanism of BN to human serum albumin.

Interaction of catecholamine precursor l-Dopa with lysozyme: A biophysical insight.

The current study comprises of an inclusive biophysical study, enlightening the binding of L-3, 4-dihydroxyphenylalanine (l-Dopa) with human lysozyme (HL) and hen egg white lysozyme (HEWL). Spectroscopic and molecular docking tools have been utilized to study the interaction of l-Dopa with both HL and HEWL. Spectrofluorimetric measurements exhibited that l-Dopa quenched the HL and HEWL intrinsic fluorescence. A binding constant (Kb) of ∼104M-1 for both HL and HEWL was obtained, asserting a significant binding. Negative value of ΔG affirmed that the reaction between proteins and l-Dopa was spontaneous. Far-UV CD spectra revealed a boost to the proteins helical content in the presence of l-Dopa. Furthermore, DLS measurements displayed the decrease in hydrodynamic radii (Rh) of HL and HEWL in the presence of l-Dopa. Molecular docking studies established that l-Dopa formed complexes with both the proteins through hydrogen bonding and hydrophobic interaction. The present study characterizing the l-Dopa interaction with lysozyme could be noteworthy in realizing both pharmaco-dynamics and/or -kinetics of drugs used in various diseases.

Cationic surfactant mediated fibrillogenesis in bovine liver catalase: a biophysical approach.

Protein aggregation into oligomers and mature fibrils are associated with more than 20 diseases in humans. The interactions between cationic surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) with varying alkyl chain lengths and bovine liver catalase (BLC) were examined by various biophysical approaches. The delicate coordination of electrostatic and hydrophobic interactions with protein, play imperative role in aggregation. In this article, we have reconnoitered the relation between charge, hydrophobicity and cationic surfactants DTAB and TTAB on BLC at pH 7.4 and 9.4 which are two and four units above pI, respectively. We have used techniques like turbidity, Rayleigh light scattering, far-UV CD, ThT, ANS, Congo red binding assay, DLS, and transmission electron microscopy. The low concentration ranges of DTAB (0-600 µM) and TTAB (0-250 µM) were observed to increase aggregation at pH 9.4. Nevertheless, at pH 7.4 only TTAB was capable of inducing aggregate. DTAB did not produce any significant change in secondary structure at pH 7.4 suggestive of the role of respective charges on surfactants and protein according to the pI and alkyl chain length. The morphology of aggregates was further determined by TEM, which proved the existence of a fibrillar structure. The surfactants interaction with BLC was primarily electrostatic as examined by ITC. Our work demystifies the critical role of charge as well as hydrophobicity in amyloid formation.

Probing the interaction of cephalosporin antibiotic-ceftazidime with human serum albumin: A biophysical investigation.

The fate of drug administered to a living organism depends on drug's pharmacokinetics as well as pharmacological behavior. Serum albumins (proteins in blood plasma of human) act as a carrier molecule to deliver the drug at specific site. In the present study, we have explored the mechanism of interaction between cephalosporin antibiotic-ceftazidime (CFD) and human serum albumin (HSA) by spectroscopic and molecular docking studies. Quenching of HSA fluorescence by CFD inferred that it binds to HSA through static quenching mechanism; with binding affinity in order of 104M-1. Fluorescence resonance energy transfer (FRET) results shows that donor and acceptor molecule are at 2.08nm apart and also reflects the high probability of energy transfer between HSA and CFD. Change in secondary structure as well as microenvironment around both tryptophan and tyrosine residue, were monitored by Circular Dichroism (CD) and Synchronous fluorescence spectroscopy respectively; confirms that CFD increases the alpha helical secondary structure as well as altered the environment around tryptophan and tyrosine. The specific binding site of CFD on HSA was determined by site-specific markers and molecular docking methods. CFD preferably bind to subdomain IIIA (Sudlow site II) on HSA.

Anti-Parkinsonian L-Dopa can also act as anti-systemic amyloidosis-A mechanistic exploration.

In spite of the fact that amyloid related neurodegenerative illnesses and non-neuropathic systemic amyloidosis have allured the research endeavors, as no cure has been announced yet apart from symptomatic treatment. Therapeutic agents which can reduce or disaggregate those toxic oligomers and fibrillar species have been studied with more compounds are on their way. The current research work describes comprehensive biophysical, computational and microscopic studies which reveal that L-3, 4-dihydroxyphenylalanine (L-Dopa) have indisputable efficacy to hinder the heat induced amyloid fibrillation of the human lysozyme (HL) and also preserve the fibril disaggregating potential. The IC50 value of L-Dopa is calculated to be 63.0±0.09µM. L-Dopa intervenes in the process of amyloid fibrillogenesis through hydrophobic interaction and hydrogen bond formation with the amino acid residues found in the amyloid fibril forming prone region of HL as clarified by molecular simulation data. L-Dopa also disaggregates the mature amyloid fibrils into some unorganized species and the DC50 value was estimated to be 19.95±0.063µM. Hence, L-Dopa and related compounds can act as effective inhibitors in the therapeutic development to combat systemic amyloidosis.

A multitechnique approach to probe the interaction of a therapeutic tyrosine kinase inhibitor nintedanib and bovine serum albumin.

Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern-Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern-Volmer equation at 3 temperatures was realized to be of the order of ~104 M-1. Negative ΔG (~-5.93 kcal mol-1), ΔH (-3.74 kcal mol-1), and ΔS (-1.50 kcal mol-1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein's symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3 nm) as obtained by the dynamic light scattering measurement results.

Polyols (Glycerol and Ethylene glycol) mediated amorphous aggregate inhibition and secondary structure restoration of metalloproteinase-conalbumin (ovotransferrin).

Under physical or chemical stress, proteins tend to form aggregates either highly ordered (amyloid) or unordered (amorphous) causing many pathological disorders in human and loss of proteins functionality in both laboratory conditions and industries during production and storage at commercial level. We investigated the effect of increasing temperature on Conalbumin (CA) and induced aggregation at 65°C. The enhanced Thioflavin T (ThT) and ANS (1-anilinonaphtalene 8-sulfonic acid) fluorescence intensity, show no shift on Congo red binding, additionally, transmission and scanning electron microscopy (TEM) (SEM) reveal amorphous morphology of the aggregate. Our investigation clearly demonstrated that polyols namely Glycerol (GL) and Ethylene glycol (EG) are so staunch to inhibit amorphous aggregates via restoring secondary conformation. Addition of polyols (15% GL and 35% EG) significantly decrease the turbidity, Rayleigh scattering ThT and ANS fluorescence intensity. The dynamic light scattering (DLS) data show that hydrodynamic radii (Rh) of the aggregates is ∼20 times higher than native CA while nearly similar for GL and EG protected CA due to condensation of core size with little difference.

A biophysical and computational study unraveling the molecular interaction mechanism of a new Janus kinase inhibitor Tofacitinib with bovine serum albumin.

The interaction of a recently certified kinase inhibitor Tofacitinib (TFB) with bovine serum albumin (BSA) has been studied, by spectroscopic and molecular docking studies. Spectrofluorimetric measurements at 3 different temperatures (288, 298, and 310 K) showed that TFB quench the intrinsic fluorescence of BSA upon forming a nonfluorescent complex. The intrinsic fluorescence data showed that TFB binds to BSA with binding constant (Kb ) of approximately 104 M-1 , affirming a significant affinity of TFB with BSA. The decrease in Stern-Volmer quenching constant with increasing temperature exhibited the static mechanism of quenching. Negative value of ΔG (-6.94 ± 0.32 kcal·mol-1 ), ΔH (-7.87 ± 0.52 kcal·mol-1 ), and ΔS (-3.14 ± 0.42 cal·mol-1 ·K-1 ) at all 3 temperatures declared the reaction between BSA and TFB to be spontaneous and exothermic. Far-UV circular dichroism spectroscopy results demonstrated an increase in helical content of BSA in the presence of TFB. Moreover, dynamic light scattering measurements showed that TFB resulted into a decrease in the hydrodynamic radii (from 3.6 ± 0.053 to 2.9 ± 0.02 nm) of BSA. Molecular docking studies confirmed that TFB binds near site II on BSA, hydrogen bonding, and hydrophobic interaction were involved in the BSA-TFB complex formation. The present study characterizing the BSA-TFB interaction could be significant towards gaining an insight into the drug pharmacokinetics and pharmacodynamics and also in the direction of rational drug designing with better competence, against emerging immune-mediated diseases, ie, alopecia and rheumatoid arthritis.