Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus

Objective To explore RNA dependent RNA polymerase of Chikungunya virus (CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen (HLA) classes as targets for epitopes based CHIKV vaccine. Methods In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases (NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. Propred I and Propred software were used to predict HLA I and HLA II binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLA I binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database (IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier. Results Twenty two HLA class II epitopes and eight HLA class I epitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486–494 and VRRLNAVLL at 331–339 were found to bind with 37 and 36 of the 51 HLA class II alleles respectively. Epitope MANRSRYQS at position 58–66 and epitopes YQSRKVENM at positions 64–72 were predicted to bind with 12 and 9 HLA II alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLA I alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection. Conclusions Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus

OBJECTIVE@#To explore RNA dependent RNA polymerase of Chikungunya virus (CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen (HLA) classes as targets for epitopes based CHIKV vaccine.@*METHODS@#In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases (NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. Propred I and Propred software were used to predict HLA I and HLA II binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLA I binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database (IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier.@*RESULTS@#Twenty two HLA class II epitopes and eight HLA class I epitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486-494 and VRRLNAVLL at 331-339 were found to bind with 37 and 36 of the 51 HLA class II alleles respectively. Epitope MANRSRYQS at position 58-66 and epitopes YQSRKVENM at positions 64-72 were predicted to bind with 12 and 9 HLA II alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLA I alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection.@*CONCLUSIONS@#Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

Determination of S- and R-warfarin enantiomers by using modified HPLC method

Warfarin is a commonly prescribed anticoagulant existing in two enantiomeric forms S- and R-warfarin. Many techniques have been used to analyze warfarin in plasma but less frequently for enantiomeric analysis. One of the HPLC method employed was further simplified and made economical. Method was validated according to ICH guidelines and was found to be sensitive, reliable and less time consuming. For both enantiomers, LLOQ was 12.5ng/mL. The CV% and accuracy for method were in the range of 0.8-14.6% and 92-107% respectively. The recoveries for both enantiomers were in the range of 86-103.8%. Blood samples were collected from 170 stable patients taking warfarin and S- and R-warfarin levels were determined by this method. Majority of subjects were found to have S/R-warfarin ratio of about 1:2 as reported in previous studies due to rapid clearance of S-enantiomer than R-enantiomer. However individual subjects data was suggestive of presence of slow metabolizers of S-warfarin leading to altered S/R ratio. Previous studies have also pointed out CYP2C9 polymorphism being responsible for such inter-individual differences in S-warfarin metabolism. So plasma warfarin S/R ratio may serve as a useful phenotypic test for CYP2C9 polymorphism

Frequency of different disorders requiring warfarin therapy and its outcome in terms of dosage and INR value in Pakistani population

To determine the frequency of different disorders requiring warfarin therapy and to see the target INR and warfarin dose requirement in Pakistani population. Descriptive study. The study was carried out at Armed Forces Institute of Cardiology [AFIC] Rawalpindi, Military Hospital Rawalpindi and National Institute of Cardiovascular Diseases [NICVD], Karachi, Pakistan from October 2010 to March 2012. Stable patients taking warfarin therapy were recruited after detailed medical history, physical examination and laboratory tests. The demographic and clinical data of individuals were entered in a pre-structured proforma. Patients suffering from hepatic and renal disease, any co-morbid disease or taking any concurrent medication or diet which would have affected warfarin therapy, were excluded. Data was analyzed using SPSS version 20.0. A total of 607 stable patients fulfilling the eligibility criteria, participated in the study. There were 297 [48.9%] male and 310 [51.1%] female patients. The mean age was 37.93 +/- 12.23 years [range 18-65 years]. The most common indication for warfarin therapy was valvular heart diseases [93.4%] followed by atrial fibrillation [2.3%] whereas other indications for warfarin use are less commonly seen in our study population. Patients had mean international normalized ratio [INR] value of 2.3 +/- 0.8 [range 1.5-3.5]. Mean daily dose of warfarin calculated in 607 patients was 5.62 +/- 1.98 mg with the range of 0.36-15 mg whereas mean weekly dose was 39.36 +/- 13.8 mg with the range of 2.5-105 mg. In Pakistani population the most common indications for warfarin use are valvular heart diseases followed by atrial fibrillation. The mean INR values were within recommended range of 2-3. The mean daily dose observed in long-term therapy is comparable to the empirical dose of 5 mg routinely started in clinical practice

Comparative bioavailability and pharmacokinetics of two oral formulations of flurbiprofen: a single-dose, randomized, open-label, two-period, crossover study in Pakistani subjects

Comparative bioavailability studies are conducted to establish the bioequivalence of generic formulation with that of branded reference formulation, providing confidence to clinicians to use these products interchangeably. This study was carried out to compare a locally manufactured formulation of flurbiprofen with that of a branded product. Twenty two healthy male adults received a single dose of flurbiprofen [100mg] either generic or branded product according to randomization scheme on each of 2 periods. Blood samples were collected and plasma flurbiprofen concentration was determined by a validated HPLC method. Pharmacokinetic parameters like AUC[0-t], AUC[0- Infinity], C[max], T[max], t[1/2] Vd and clearance were determined. The 90% CI for the ratio of geometric means of test to reference product's pharmacokinetic variables was calculated. Pharmacokinetic parameters for two formulations were comparable. Ratio of means of AUC[0-24], AUC[0- Infinity] and C[max] for test to reference products and 90% CI for these ratios were within the acceptable range. The p-values calculated by TOST were much less than the specified value [p-0.05]. ANOVA gave p-values which were more than the specified value [p-0.05] for sequence, subject, period and formulation. Test formulation of flurbiprofen [tablet Flurso] was found to meet the criteria for bioequivalence to branded product [tablet Ansaid] based on pharmacokinetic parameters

Role of relaxant prostaglandins in the effect of nebivolol on isolated tracheal muscle of guinea pig

The present study was therefore aimed to evaluate the role of relaxant prostaglandins in modulating the effect of Nebivolol on tracheal muscle of guinea pig since the exact mechanism underlying its effects on tracheal muscle has not been established yet. The use of beta blockers is limited by their ability to produce bronchospasm in asthmatics. Third generation beta blockers like Nebivolol may show better tolerability since there may be involvement of relaxant prostaglandins in its effect. However the involvement of prostaglandins in the respiratory effects of Nebivolol remains unexplored. The present study, carried out on isolated tracheal muscle strips of guinea pigs was designed to explore this controversy. Experimental Study. This study was conducted at the department of Pharmacology, Army Medical College, Rawalpindi since April 2010 to November 2010. varying concentration of histamine ranging from 10[-7]M to 10[-3]M were used to plot a concentration response curve on the isolated tracheal muscle strips of guinea pig and was used as a control. The same concentration response curve was plotted in presence of a fixed concentration of Nebivolol 10[-6]M then again in presence of a fixed concentration of Indomethacin 10[-6]M and Nebivolol 10[-6]M together in a series of experiments using six sets of isolated tracheal muscle strips in each case. Nebivolol did not produce any significant shift in the concentration response curve in the presence and absence of Indomethacin. Nebivolol does not augment the histamine induced contraction of respiratory smooth muscle of guinea pig in the presence of Indomethacin, prostaglandin synthesis inhibitor indicating no role of relaxant prostaglandins in the sparing of respiratory smooth muscle by Nebivolol

Pharmacokinetic profile of chloroquine in healthy Pakistani subjects

To study pharmacokinetics of chloroquine in healthy adult Pakistani subjects and evaluate the role of pharmacokinetic factor in the dynamics of drug resistance in Plasmodium falciparum. This was an experimental study. The study was conducted at National Institute of Health Islamabad during the year 2001. Eleven healthy male volunteers, who met the defined inclusion criteria, were recruited into the study after obtaining informed consent. Blood samples were drawn at specified time intervals before and after administration of chloroquine tablets equivalent to 600 mg base. Plasma was separated and extracted according to the methods described in literature. Chloroquine estimation was carried out using High Performance Liquid Chromatography. Data of plasma concentration of chloroquine in 10 healthy volunteers is reported in this study. Mean pharmacokinetic parameters worked out from the data are Area under the Curve = 10.827, Peak Concentration = 201 ng/ml, Time to Peak = 6.1 hours, Half Life Phase-1 = 2.62 hours, Half Life Phase-2 = 41 hours, Volume of Distribution = 149.24 Litres, Clearance = 55.95 litres/hour Mean Residence Time = 58.53 hours. The pharmacokinetic profile of chloroquine in healthy adult male Pakistani subjects is comparable to that described in other ethnic populations of the world. The pharmacokinetic factor may play a role in selection of partially chloroquine-resistant parasites when the drug is used for prophylaxis of malaria on weekly basis

Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase

The free radical scavenging activity of ethanolic and aqueous extracts from six plants has been evaluated, in vitro, using 1, 1-diphenyl-2-picrylhydrazyl [DPPH] radical scavenging method and compared with ascorbic acid, quercetin. kaempferol and apigenin. Extracts of Pistacia integerrima leaves and galls exhibited highest DPPH radical scavenging activity among all the plants extracts examined. Carissa carandas from Apocynaceae and Vitex negundo of Verbenaceae family also demonstrated DPPH radical scavenging activity. Inhibitory effects towards the in-vitro reaction of hypoxanthine and xanthine oxidase [XO] was also carried out in the presence of plant extract, aglycones quercetin, kaempferol and apigenin along with allopurinol The extracts from Pistacia integerrima galls exhibited highest XO inhibitory activity than leaves of Pistacia integerrima and other plant extracts, was comparable with aglycones but less than allopurniol, a pharmacologically different structure. The increased DPPH radical scavenging and XO inhibitors activity by Pistacia integerrima galls may be partly exerted by flavonoids produced during stress of an insect aphids [Pemphogus species] attack on the leaves. The results indicate that Pistacia integerrima may be a good candidate for future studies in vivo models of gout and hyperuricemia