Nano crystalline cellulose based drug delivery system for some non-steroidal anti-inflammatory drugs: Synthesis, characterization and in-vitro simulation studies.

Nano crystalline cellulose (NCC) created a breakthrough in biomedical field because of its important characteristics like large surface area, good mechanical strength, biocompatibility, renewability and feasibility of incorporation to both hydrophilic and hydrophobic substances. In the present study NCC based drug delivery systems (DDSs) of some non-steroidal anti-inflammatory drugs (NSAIDs) were obtained by covalent bonding between hydroxyl groups of NCC with carboxyl group of NSAIDs. Developed DDSs were characterized by means of FT-IR, XRD, SEM and thermal analysis. In-vitro release study and fluorescence study showed that these systems are stable up to 18 h in upper gastrointestinal (GI) tract at pH 1.2 and released NSAIDs in sustained manner over the period of 3 h in intestine at pH 6.8-7.4. Present study performed with the aim to reuse bio-waste even in the form of DDSs is of greater therapeutic efficacy with reduced dosing frequency that overcome physiological adversities involved with NSAIDs.

Genome-Wide Association Study and Post-genome-Wide Association Study Analysis for Spike Fertility and Yield Related Traits in Bread Wheat.

Spike fertility and associated traits are key factors in deciding the grain yield potential of wheat. Genome-wide association study (GWAS) interwoven with advanced post-GWAS analysis such as a genotype-phenotype network (geno-pheno network) for spike fertility, grain yield, and associated traits allow to identify of novel genomic regions and represents attractive targets for future marker-assisted wheat improvement programs. In this study, GWAS was performed on 200 diverse wheat genotypes using Breeders' 35K Axiom array that led to the identification of 255 significant marker-trait associations (MTAs) (-log10P ≥ 3) for 15 metric traits phenotyped over three consecutive years. MTAs detected on chromosomes 3A, 3D, 5B, and 6A were most promising for spike fertility, grain yield, and associated traits. Furthermore, the geno-pheno network prioritised 11 significant MTAs that can be utilised as a minimal marker system for improving spike fertility and yield traits. In total, 119 MTAs were linked to 81 candidate genes encoding different types of functional proteins involved in various key pathways that affect the studied traits either way. Twenty-two novel loci were identified in present GWAS, twelve of which overlapped by candidate genes. These results were further validated by the gene expression analysis, Knetminer, and protein modelling. MTAs identified from this study hold promise for improving yield and related traits in wheat for continued genetic gain and in rapidly evolving artificial intelligence (AI) tools to apply in the breeding program.

Nano-crystalline cellulose: Preparation, modification and usage as sustained release drug delivery excipient for some non-steroidal anti-inflammatory drugs.

Nano crystalline cellulose (NCC) has gained attention in the pharmaceutical industry owing to its biodegradable nature and high mechanical properties. In the present work we reported preparation of NCC from cellulose isolated from a bio-waste, its modification and exploration as drug delivery excipient for sustained release of non-steroidal anti-inflammatory drugs (NSAIDs). Cellulose extracted from Citrus limetta albedo was subjected to alkali treatment, bleaching and hydrolysis by sulfuric acid to obtain NCC of particle size ranging from 1 to 10 nm. Further, modification with a cationic surfactant cetyltrimethylammonium bromide (CTAB) resulted in NCC-CTAB with enhanced surface area and high aspect ratio which was investigated as sustained release drug delivery system (DDS) for NSAID. Drug loading capacity correlated well with log P value of NSAID. NSAID-loaded NCC-CTAB acted as sustained release DDS over a period of 3hr. The results were confirmed as serum protein protecting and anti-cathepsins activities. CTAB-NSAID interactions have been assessed using CHEM3D.

N-formylpyrazolines and N-benzoylpyrazolines as novel inhibitors of mammalian cathepsin B and cathepsin H.

Cathepsins, intracellular proteases, are known to be involved in a number of physiological processes ranging from degradation of extracellular proteins, prohormone processing, progressions of atherosclerosis, etc. High levels of cathepsins have been indicated in various pathological conditions like arthritis, cancer and other tissue degenerative disorders. One of the reasons attributed to these high levels is decrease in inhibitor concentration. Therefore, the work on the identification of small molecular weight compounds as inhibitors of cysteine proteases is of great therapeutic significance. Exploring this work in the same direction, we here present the synthesis of substituted N-formylpyrazolines and N-benzoylpyrazolines and study these as inhibitors to cysteine proteases. After a preliminary screening of the compounds as inhibitors to cysteine proteases in general, studies were carried out to study their inhibitory effects on cathepsin B and cathepsin H. SAR studies show that N-formylpyrazolines were better inhibitors than N-benzoylpyrazolines. The most potent inhibitors among the two series were nitro substituted compounds 1i and 2i with Ki values of ∼1.1×10(-9)M and 19.5×10(-8)M for cathepsin B and Ki values of ∼5.19×10(-8)M and 9.8×10(-7)M for cathepsin H, respectively. Docking experiments showing interaction between N-formylpyrazolines and N-benzoylpyrazolines with enzyme active sites structures also provided useful insights.

SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of cathepsin B and cathepsin H.

Cathepsins have emerged as a potential target for anti-cancer drug development. In the present study, we have synthesized three structurally related series of flavanoids i.e., 2'-hydroxychalcones, flavanones and flavones and assayed in vitro to study their inhibitory potency against cathepsin B and H, promising drug candidate for cancer therapy. Enzyme kinetics studies were carried out in presence of these compounds after preliminary proteolytic studies on endogenous protein substrates. SAR studies suggested that open chain flavanoids were better inhibitors as compared to their cyclized analogs. The most potent inhibitors among the three series were nitro substituted compounds 1g, 2g and 3g with Ki values of ∼6.18×10(-8) M, 4.8×10(-7) M and 7.85×10(-7) M for cathepsin B and Ki values of ∼2.8×10(-7) M, 31.8×10(-6) M and 33.7×10(-6) M for cathepsin H, respectively. The relationship between chalcone, flavanones and flavone structures interpreted by docking studies on cathepsin B and H also provided useful insights.

Case report: Report of a rare case of juvenile recurrent parotitis and review of literature.

BACKGROUND: Juvenile recurrent parotitis (JRP) is a rare salivary gland disease of obscure aetiology that affects children. It is characterized by multiple episodes of unilateral or bilateral parotid inflammation over a period of years. CASE REPORT: A 14 year old boy presented with multiple episodes of recurrent bilateral swellings of the parotid glands since 1 year of age with no relevant past medical and dental history, TREATMENT: Included prescription of antibiotic Dicloxacillin 500 mg tid for 7 days and analgesics as a combination of Diclofenac 50 mg and Paracetamol 500 mg tid for 10 days, to resolve acute infection followed by sialography using Iopromide (ultravist-300) twice at an interval of 6 months for glandular lavage which helps to clear the mucous plugs that form during the acute phase. FOLLOW-UP: It was satisfactory as there has been no recurrence of parotitis during 18 months.

Physico-chemical properties of brain cathepsin H.

Cathepsin H (EC 3.4.22.16) from cow brain, purified to approximately 1800-fold with approximately 26% activity yield, hydrolysed BANA, Leu-2-NNap, Arg-2-NNap, and Met-2-NNap maximally at pH 6.5, 6.8, 7.0 and 7.2, respectively. It was activated by sulphydryl compounds and EDTA while sulphydryl alkylators and blockers were found to inhibit the enzyme activity. Met-2-NNap was found to be the best substrate followed by Thr-2-NNap, His-2-NNap, Leu-2-NNap, Arg-2-NNap and Ala-2-NNap, respectively. The Km values for hydrolysis of various substrates viz., Met-2-NNap, Leu-2-NNap, Arg-2-NNap, Arg-NNapOMe, Thr-2-NNap, His-2-NNap, BANA, Arg-pNA and Lys-pNA were 0.128, 0.167, 0.169, 0.288, 0.428, 0.500, 0.667, 0.195 and 0.476 mM, respectively. The temperature optima for hydrolysis of BANA and Leu-2-NNap were approximately 45 degrees C and approximately 50 degrees C with activation energies of approximately 13.7 and approximately 11.0 kcal mole-1, respectively. The enzyme was fairly stable upto 50 degrees C and between pH 4.0-7.5.

Effect of some steroidal & non-steroidal anti-inflammatory drugs on purified goat brain cathepsin L.

The lysosomal cysteine proteinases have been found to be involved in various inflammatory conditions. Inhibitory effects of certain commonly used anti-inflammatory drugs were observed on lysosomal thiol proteinase cathepsin L. Of the non-steroidal anti-inflammatory drugs tested, phenylbutazone was found to be most potent inhibitor of cathepsin L activity. The half maximal inhibition was achieved at 0.6 mM concentration. The inhibition by phenylbutazone was of non-competitive type, with a ki of 1.3 x 10(-3) M. Flufenamic acid and indomethacin were also inhibitory to cathepsin L activity, giving half maximal inhibitions at 3.5 mM and 4.5 mM concentrations respectively. In contrast, cathepsin L activity was not affected at all by steroidal anti-inflammatory agents. Aspirin was also found to have no effect on cathepsin L activity whereas salicylic acid, its m- and p-analogs exhibited inhibitory effects but to a lesser degree.

Effect of some pesticides/weedicides on cathepsin B activity and lysosomal membrane.

The in vitro inhibitory effects of various weedicides and pesticides on goat brain cathepsin B and their labilizing potency on the lysosomal membrane were quantitated. Endosulfan an organochlorine insecticide inhibited the enzymic activity to approximately 50% at 7 mM concentration followed by methyl parathion, aldrin, melathion and benzene hexachloride (BHC) in that order. Among the weedicides, butachlor was found to be most inhibitory (approximately 50% activity was lost at 6 mM) followed by isoproturone (28%) and anilophos (19%). When the labilizing/stabilizing potency of all these drugs was observed on lysosomal membrane it was found that none of these was capable of stabilizing the membrane. At 40 degrees C and 1 mM drug concentration, aldrin, endosulfan, melathion and anilophos were found to be strong labilizers of the lysosomal membrane. Others like isoproturone, BHC and methyl parathion had moderate labilizing effect. The labilization potency of the drugs was temperature dependent and was less pronounced at 25 degrees C as compared to 40 degrees C.

Dipeptidyl peptidase I from goat brain: purification, characterization and its action on Leu-enkephalin.

Brain dipeptidyl peptidase (DPP) I has been purified 2990-fold to apparent homogeneity shown by a single protein band in electrophoreses at pH 4.5, 8.4 and in SDS-PAGE at pH 7.2. The purification techniques included homogenization of brain acetone powder, autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation. Sephadex G-100 column chromatography, heat treatment at 65 C. organomercurial affinity chromatography. CM-Sephadex cation-exchange chromatographies at pH 5.6 and 5.0 and anion-exchange chromatography on DEAE-Sephadex at pH 6.8. The enzyme hydrolysed synthetic substrate Gly-Arg-4-methoxy-beta-naphthyl-amide maximally at pH 6.0. The Km values for Gly-Arg-beta-naphthylamide and Gly-Arg-4-methoxy-beta-naphthylamide substrates were 0.10 mM and 0.14 mM respectively. The enzyme was inhibited by thiol inhibitors like p-chloromercuribenzoic acid, iodoacetic acid, iodoacetamide and microbial inhibitors leupeptin and antipain. Molecular weight estimations on a calibrated Sephadex G-200 column afforded a value of 180,000 Da while in denaturing conditions on sodium dodecyl sulphate polyacrylamide gel electrophoresis, the subunit molecular weight was 22,000 Da. The subunit structure of the native enzyme was unfolded in presence of different concentrations of urea. In 8 M urea, the enzyme dissociated completely into monomers of 25,000 Da but 6, 5 and 4 M urea concentrations revealed the existence of dimers, tetramers and hexamers. Leu-enkephalin. Tyr-Gly-Gly-Phe-Leu was degraded by DPPI into Tyr-Gly and Gly-Phe-Leu with no further degradation of the newly generated tripeptide.

A selective colorimetric assay for cathepsin L using Z-Phe-Arg-4-methoxy-beta-naphthylamide.

Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.