Formulation And Development Of Gastroretentive Drug Delivery System Of Efavirenz

Efavirenz, a non-nucleotide reverse transcriptase inhibitor is an important drug for treating patients with Human Immunodeficiency Virus infections. It belongs to BCS class II have low solubility and poor intrinsic dissolution rate. It is highly basic (pKa 10.2) which makes it suitable candidate for floating dosage form for continuous delivery in stomach.The study was aimed to improve the solubility by solid dispersion technique.Saturation solubility study and drug content were evaluated for solid dispersion preparation. Saturation solubility shows 8 fold increases in 0.1 N HCL compared to plain drug and drug content was found to be between 95%-102%. Further effervescent floating gastroretentive drug delivery system was prepared by 32 full factorial design with independent variables i.e., concentration of HPMC K100 as matrix forming agent and citric acid as gas generating agent. Lag time, floating time, percent drug release were studied as responses. The optimized batch exhibited floating lag time of 40 sec and the in vitro release studies showed 89.5% drug release in 9 h and tablet remained floating for greater than 8 h. The study thus demonstrated that solubility is increased by solid dispersion technique and floating delivery systems may increase solubility and bioavailability of Efavirenz.

Multiparticulate Drug Delivery System For Gastrointestinal Tuberculosis

Drug solubility poses numerous challenges in design of formulations for drugs with poor aqueous solubility. Ethionamide is an antitubercular drug belonging to biopharmaceutical classification system class II drug having less aqueous solubility. Nanosuspensions were prepared by using various solvents such as methanol, ethanol, acetone and chloroform and it was prepared using anti-solvent precipitation technique by using probe sonication. Various stabilizers such as tocopherolpolythytlene glycol succinate, polyvinylpyrrolidone and tween 80 singly or in combination were studied. A 32 factorial design was employed to study the effect of independent variables, concentration of stabilizers and stirring speed on particle size and cumulative percent drug release. The particle size of the optimized batch was 97.54 ± 8.47 nm with polydispersity index of 0.36 and zeta potential -10.1 ± 2.3 mV. The cumulative percent drug release of optimized batch was found to be 95.01 ± 1.16% in 60 min. Optimized batch was ultracentrifuged and evaluated for saturation solubility studies, stability and powder X-ray Diffraction studies. Optimized nanosuspension was loaded on Espheres by spraying in a coating pan and then coating of Eudragit controlled release polymers. The coated Espheres were evaluated for drug content, friability, scanning electron microscopy, ex-vivo permeation studies and drug release kinetics studies. The friability value for primary coated sphere was found to be 0.8 ± 0.12% and for secondary was 1% and the best fit model was found to be Korsmeyer-Peppas model which is indicative of diffusion controlled release. Ex vivo diffusion studies revealed a moderate increase in permeability.

Prevalence of KRAS mutations in metastatic colorectal cancer: A retrospective observational study from India.

BACKGROUND: One of the genetic alterations implicated in tumor progression in colorectal cancers (CRCs) are abnormalities in Kristen Rat Sarcoma (KRAS) gene. Evaluation of KRAS mutation status is an important prognostic factor and has predictive value in deciding first line therapy based on monoclonal antibodies such as Cetuximab and Panitumumab in metastatic CRCs. MATERIALS AND METHODS: In this retrospective study, we analyzed 7 different somatic mutations in Exon 2 of KRAS gene in 299 unselected incidental CRC patients who visited the hospital for clinical management during the period 2009–2013. Most of the tumors were primarily originating from colon and rectum; nevertheless, there were a few from rectosigmoid, sigmoid, ceacum and anal canal in the study group. Genomic DNA extracted from paraffin embedded tumor tissues was screened for 7 point mutations located in Codons 12 and 13 of KRAS gene, using Scorpions amplified refractory mutation system real time polymerase chain reaction technology. Statistical analysis was performed to assess bivariate relationship between different variables that includes: mutation status, mutation type, tumor location, tumor morphology, age and sex. RESULTS: Prevalence of mutation in Codons 12 and 13 was 42.8% in the study group. Well‑differentiated tumors had significantly more mutation positivity than moderately and poorly differentiated tumors (P = 0.001). 92% of the mutations were from Codon 12 and 8% in Codon 13. Glycine to Arginine was relatively more common in rectosigmoid followed by ceacum, while Glycine to Alanine mutation was relatively more prevalent in sigmoid, followed by rectum and rectosigmoid. CONCLUSION: The results suggest a prevalence of KRAS mutation at 42.8% in Indian population indicating that this testing is very crucial for targeted therapy management in metastatic CRC in India. Further analysis on mutation status of other homologues such as NRAS and downstream partner, v‑raf murine sarcoma viral oncogene homolog B1, would add value to understanding the role of anti‑epidermal growth factor receptor therapy in CRC management.

Fructose, 1,6-diphosphate mitigates CCl4 suppressed nitric oxide synthase activity in rats.

Effect of fructose 1,6-diphosphate (FDP) and carbon tetrachloride (CCl4) were studied individually and in combination on rat endothelial (ET) and smooth muscle cell (SMC) nitric oxide synthase (NOS) activities in vivo, inhibition of ET and SMC NOS activity in CCl4 treated rats was reversed in FDP + CCl4 treated animals. Cellular based NOS activity was significantly increased in FDP treated group of rats when compared to non treated controls. The results suggest a significant increase in NOS in rats treated with a combination of FDP + CCl4 thus overcoming the suppression of NOS exposed to CCl4 alone.

Suitability of CANSCORE for the assessment of the nutritional status of newborns.

The suitability of Clinical Assessment of Nutritional Status Score (CANSCORE) for the assessment of foetal malnutrition among 372 local Hyderabad newborns was studied. Details of length, weight and body mass index (BMI) at birth were related to total CANSCORE which consisted of scores ranging from 1 to 4 based on the grades of clinical status of hair, cheeks, buttocks, chest, legs, back, neck, arms and skin of anterior abdominal wall. The correlation coefficients of CANSCORE with the length, weight and BMI of newborns indicated that score of hair was least correlated with nutritional status. Normal newborns were found to have the lowest prevalence of foetal malnutrition. In those with retarded measurements of length and weight or BMI, the prevalence of foetal malnutrition was higher. The newborns with retardation of both length and BMI had higher prevalence of foetal malnutrition. The feasibility of the suggested limits of CANSCORE for the foetal malnutrition was assessed. Values of 24 for total CANSCORE and of 22 for "Modified CANSCORE" (score excluding hair as a parameter) were found appropriate for the assessment of foetal malnutrition. Modified CANSCORE is a simple, rapid and quantifiable method for the assessment of foetal malnutrition in term newborns.

Inhibition of rat brain nitric oxide synthase activity by cyclosporin-A.

Cyclosporin-A, a potent immunosuppressive agent is known to induce cellular toxic side effects by way of altering calcium homeostasis, including calcium/calmodulin mediated events. We studied the in vitro and in vivo effects of cyclosporin-A on rat brain nitric oxide synthase activity (the enzyme that mediates the conversion of L-arginine to citrulline and NO). CsA in concentrations of 22-4400 nM inhibited rat brain NOS activity in vitro and in 10 or 25 mg/kg wt/4 weeks of CsA treated rat brains in vivo. We report here that CsA by way of interfering with rat brain Ca2+/CaM mediated events may inhibit its NOS activity which ultimately may result in neurotoxicity.

Cyclosporin A-induced changes in nitric oxide synthase activity in the rat kidney: in vitro and in vivo assay.

Cyclosporin A (CsA) is a cyclic undecapeptide that has been used extensively as an immunosuppressive drug in transplantation medicine and is known to interact with L-arginine dependent pathways. We studied the in vitro and in vivo effects of CsA on nitric oxide synthase (NOS) activity in the rat kidney. CsA in concentrations of 44-2200 nM in vitro, and 12.5 or 25 mg/kg body weight per 4 weeks treated rat kidneys in vivo, significantly stimulated NOS activity. CsA may alter the Ca2+/Cam-dependent NOS activity by interacting with rat kidney Ca2+/calmodulin dependent events.

Testis-specific histone (H1t) is not phosphorylated and has a weak interaction with chromatin.

Soluble chromatin was prepared from rat testes after a brief micrococcal nuclease digestion. After adsorption onto hydroxylapatite at low ionic strength, the histone H1 subtypes were eluted with a shallow salt gradient of 0.3 M NaCl to 0.7 M NaCl. Histone H1t was eluted at 0.4 M NaCl, while histones H1a and H1c were eluted at 0.43 M NaCl and 0.45 M respectively. The extreme divergence of the amino acid sequence of the C-terminal half of histone H1t, the major DNA binding domain of histone H1, from that of the somatic consensus sequence may contribute to the weaker interaction of histone H1t with the rat testis chromatin. Further, histone H1t was not phosphorylated in vivo in contrast to histone H1a and H1c, as is evident from the observation that histone H1t lacks the SPKK motif recognized by the CDC-2kinase or the RR/KXS motif recognized by protein kinase A.

Cavernous haemangioma of conjunctiva (a case report).

A rare case of Cavernous Haemangioma of the conjunctiva which was treated successfully by surgery is reported.

Effect of chemical modification on struture and activity of glucoamylase from Aspergillus candidus and Rhizopus species.

The histidine, tyrosine, tryptophan and carboxyl groups in the enzyme glucoamylase from Aspergillus Candidus and Rhizopus species were modified using group specific reagents. Treatment of the enzyme with diethylpyrocarbonate resulted in the modification of 0·3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase from Aspergillus candidus and Rhizopus species respectively. Modification of tyrosine either by Nacetylimidazole or [I125]-leads to a partial loss of activity. Under denaturing conditions, maltose did not help in protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the presence of urea, photooxidation at pH 9·0, N-bromosuccinamide at pH 4·8 resulted in a complete loss of activity· However, the results of experiments in the presence of maltose and at pH 4·8 photooxidation and Nbromosuccinamide treatment suggested the presence of two tryptophan residues at the active site. There was a complete loss of enzyme activity when 10 and 28 carboxyl groups from Aspergillus candidus and Rhizopus, respectively were modified. Modification in the presence of substrate maltose, showed at least two carboxyl groups were present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 types of α-glucosidic linkages namely α-1,4, α-1, 6 and α-l,3.

Fungal glucoamylases.

The purification and properties of glucoamylase (α-l,4-glucan glucohydrolase, EC 3.2.1.3) from different fungal sources have been compared. The studies on the conformation and activity of the native enzyme at a function of pH, temperature, substrate concentration and the effect of denaturants and on the role of carbohydrate moiety on structure and stability have been reviewed. The chemical modification of the active centre, binding kinetics of the substrate and active site and the mechanism of action have been summarized. They differ in their fine structure as revealed by their near ultra-violet circular dichroism spectra and contain 30–35 % α-helix, 24–36 % β-structure and the rest aperiodic structure. The activity of the enzyme is very sensitive to the environment around aromatic aminoacid residues. The glucoamylases are glycoprotein in nature, differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity. At the active site of the enzyme, two tryptophan and two carboxyl (glutamate or aspartate) groups are present. It is likely that the histidine and tyrosine residues which are present away from the active site are involved in binding of the substrate. There seems to be seven subsites which are involved in binding of the substrate and the catalytic site is situated in between 1 and 2 subsites. In breaking of α-1,4-, α-1,3-, and α-l,6-bonds only one active centre is involved. Studies on the immobilization of either glucoamylase alone or as a part of a multienzyme system have been reviewed briefly.

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A.

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation.

Structure and stability of glucoamylase II from Aspergillus niger: A circular dichroism study.

Glucoamylase II (EC 3.2.1.3) from Aspergillus niger has 31 % α-helix, 36 % ßstructure and rest aperiodic structure at pH 4·8 as analysed by the method of Provencher and Glockner (1981, Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4·8 than at pH 7·0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30°C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.

Studies on carbohydrate moieties of the glycoprotein, glucoamylase II of Aspergillus niger: Nature of carbohydratepeptide linkage and structure of oligosaccharides.

Electrophoretically homogeneous type 1 (GP-C1, and GP-C 2), type 2 (GP-C3a and GP-C3b,) and type 3 (GP-D1, and GP-D2) glycopeptides from Aspergillus niger glucoamylase II (Manjunath and Raghavendra Rao, preceding paper) were separately treated with alkaline borohydride. The (ß-eliminated oligosaccharides were subjected to single and sequential digestion with specific glycosidases and the products analysed by gas liquid chromatography. The studies revealed that carbohydrate moieties were present as mannose, Man-Man-, and trisaccharide structures, namely, (a) GIc-Man-Man-, (b) Gal-Man-Man, (c) Man-Man-Man-, (d) GlcNAc-Man-Man-, and (e) Xyl-Man-Man. None of the glycopeptides contained all the trisaccharide structures (a) to (e). Type 1 glycopeptide contained structures (a), (b) and (c); type 2, (a) and (d)and type 3, (a), (b)and (e). The number of carbohydrate units (mono-, di-and trisaccharides) present in the major glycopeptides was determined and tentative structures for the glycopeptides proposed. Carbohydrate units appeared to occur in clusters of 4 to 7 in each glycopeptide, a structure unique to the carbohydrate moiety in Aspergillus niger glucoamylase. Based on carbohydrate analysis and yields of glycopeptide, the number of units of each type of glycopeptide present in glucoamylase II was tentatively calculated to give two of type Man:Glc:Gal = 12-15:l:l, one of type Man:Glc:GlcN = 10-l 1:1:2 and one of type Man :GIc :Gal :Xyl =4-8:0.1:0.5-0.8:0.3-1 glycopeptides.