Crisis averted: a world united against the menace of multiple drug-resistant superbugs -pioneering anti-AMR vaccines, RNA interference, nanomedicine, CRISPR-based antimicrobials, bacteriophage therapies, and clinical artificial intelligence strategies to safeguard global antimicrobial arsenal.

The efficacy of antibiotics and other antimicrobial agents in combating bacterial infections faces a grave peril in the form of antimicrobial resistance (AMR), an exceedingly pressing global health issue. The emergence and dissemination of drug-resistant bacteria can be attributed to the rampant overuse and misuse of antibiotics, leading to dire consequences such as organ failure and sepsis. Beyond the realm of individual health, the pervasive specter of AMR casts its ominous shadow upon the economy and society at large, resulting in protracted hospital stays, elevated medical expenditures, and diminished productivity, with particularly dire consequences for vulnerable populations. It is abundantly clear that addressing this ominous threat necessitates a concerted international endeavor encompassing the optimization of antibiotic deployment, the pursuit of novel antimicrobial compounds and therapeutic strategies, the enhancement of surveillance and monitoring of resistant bacterial strains, and the assurance of universal access to efficacious treatments. In the ongoing struggle against this encroaching menace, phage-based therapies, strategically tailored to combat AMR, offer a formidable line of defense. Furthermore, an alluring pathway forward for the development of vaccines lies in the utilization of virus-like particles (VLPs), which have demonstrated their remarkable capacity to elicit a robust immune response against bacterial infections. VLP-based vaccinations, characterized by their absence of genetic material and non-infectious nature, present a markedly safer and more stable alternative to conventional immunization protocols. Encouragingly, preclinical investigations have yielded promising results in the development of VLP vaccines targeting pivotal bacteria implicated in the AMR crisis, including Salmonella, Escherichia coli, and Clostridium difficile. Notwithstanding the undeniable potential of VLP vaccines, formidable challenges persist, including the identification of suitable bacterial markers for vaccination and the formidable prospect of bacterial pathogens evolving mechanisms to thwart the immune response. Nonetheless, the prospect of VLP-based vaccines holds great promise in the relentless fight against AMR, underscoring the need for sustained research and development endeavors. In the quest to marshal more potent defenses against AMR and to pave the way for visionary innovations, cutting-edge techniques that incorporate RNA interference, nanomedicine, and the integration of artificial intelligence are currently under rigorous scrutiny.

Potential Immunogenic Activity of Computationally Designed mRNA- and Peptide-Based Prophylactic Vaccines against MERS, SARS-CoV, and SARS-CoV-2: A Reverse Vaccinology Approach.

The continued emergence of human coronaviruses (hCoVs) in the last few decades has posed an alarming situation and requires advanced cross-protective strategies against these pandemic viruses. Among these, Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) have been highly associated with lethality in humans. Despite the challenges posed by these viruses, it is imperative to develop effective antiviral therapeutics and vaccines for these human-infecting viruses. The proteomic similarity between the receptor-binding domains (RBDs) among the three viral species offers a potential target for advanced cross-protective vaccine designs. In this study, putative immunogenic epitopes including Cytotoxic T Lymphocytes (CTLs), Helper T Lymphocytes (HTLs), and Beta-cells (B-cells) were predicted for each RBD-containing region of the three highly pathogenic hCoVs. This was followed by the structural organization of peptide- and mRNA-based prophylactic vaccine designs. The validated 3D structures of these epitope-based vaccine designs were subjected to molecular docking with human TLR4. Furthermore, the CTL and HTL epitopes were processed for binding with respective human Lymphocytes Antigens (HLAs). In silico cloning designs were obtained for the prophylactic vaccine designs and may be useful in further experimental designs. Additionally, the epitope-based vaccine designs were evaluated for immunogenic activity through immune simulation. Further studies may clarify the safety and efficacy of these prophylactic vaccine designs through experimental testing against these human-pathogenic coronaviruses.

Computational Evaluation of Abrogation of HBx-Bcl-xL Complex with High-Affinity Carbon Nanotubes (Fullerene) to Halt the Hepatitis B Virus Replication.

Hepatitis B virus (HBV) is the world's most prevalent chronic viral infection. More than 350 million individuals are chronic carriers of the virus, with an estimated 2 billion infected persons. For instance, the role of HBx protein in attachment and infection is very obvious and consequently deemed as an important druggable target. Targeting the interface and discovering novel drugs greatly advanced the field of therapeutics development. Therefore, in the current study, HBx to Bcl-xL is abrogated on high-affinity carbon nanotubes using computational structural biology tools. Our analysis revealed that among the total 62 carbon fullerenes, only 13 compounds exhibited inhibitory activity against HBx, which was further confirmed through IFD-based rescoring. Structural dynamics investigation revealed stable binding, compactness, and hydrogen bonds reprogramming. Moreover, the binding free energy calculation results revealed that the top hits1-4 possess the total binding energy of -54.36 kcal/mol (hit1), -50.81 kcal/mol (hit2), -47.09 kcal/mol (hit3), and -45.59 kcal/mol for hit4. In addition, the predicted KD values and bioactivity scores further validated the inhibitory potential of these top hits. The identified compounds need further in vitro and in vivo validation to aid the treatment process of HBV.

Rational design of potent anti-COVID-19 main protease drugs: An extensive multi-spectrum in silico approach.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a novel coronavirus and the etiological agent of global pandemic coronavirus disease (COVID-19) requires quick development of potential therapeutic strategies. Computer aided drug design approaches are highly efficient in identifying promising drug candidates among an available pool of biological active antivirals with safe pharmacokinetics. The main protease (MPro) enzyme of SARS-CoV-2 is considered key in virus production and its crystal structures are available at excellent resolution. This marks the enzyme as a good starting receptor to conduct an extensive structure-based virtual screening (SBVS) of ASINEX antiviral library for the purpose of uncovering valuable hits against SARS-CoV-2 MPro. A compound hit (BBB_26580140) was stand out in the screening process, as opposed to the control, as a potential inhibitor of SARS-CoV-2 MPro based on a combined approach of SBVS, drug likeness and lead likeness annotations, pharmacokinetics, molecular dynamics (MD) simulations, and end point MM-PBSA binding free energy methods. The lead was further used in ligand-based similarity search (LBSS) that found 33 similar compounds from the ChEMBL database. A set of three compounds (SCHEMBL12616233, SCHEMBL18616095, and SCHEMBL20148701), based on their binding affinity for MPro, was selected and analyzed using extensive MD simulation, hydrogen bond profiling, MM-PBSA, and WaterSwap binding free energy techniques. The compounds conformation with MPro show good stability after initial within active cavity moves, a rich intermolecular network of chemical interactions, and reliable relative and absolute binding free energies. Findings of the study suggested the use of BBB_26580140 lead and its similar analogs to be explored in vivo which might pave the path for rational drug discovery against SARS-CoV-2 MPro.

A computational study to disclose potential drugs and vaccine ensemble for COVID-19 conundrum.

The nucleocapsid (N) protein of SARS-COV-2, a virus responsible for the current COVID-19 pandemic, is considered a potential candidate for the design of new drugs and vaccines. The protein is central to several critical events in virus production, with its highly druggable nature and rich antigenic determinants making it an excellent anti-viral biomolecule. Docking-based virtual screening using the Asinex anti-viral library identified binding of drug molecules at three specific positions: loop 1 region, loop 2 region and ß-sheet core pockets, the loop 2 region being the most common binding and stable site for the bulk of the molecules. In parallel, the protein was characterized by vaccine design perspective and harboured three potential B cell-derived T cell epitopes: PINTNSSPD, GVPINTNSS, and DHIGTRNPA. The epitopes are highly antigenic, virulent, non-allergic, non-toxic, bind with good affinity to the highly prevalent DRB*0101 allele and show an average population coverage of 95.04%. A multi-epitope vaccine ensemble which was 83 amino acids long was created. This was highly immunogenic, robust in generating both humoral and cellular immune responses, thermally stable, and had good physicochemical properties that could be easily analyzed in in vivo and in vitro studies. Conformational dynamics of both drug and vaccine ensemble with respect to the receptors are energetically stable, shedding light on favourable conformation and chemical interactions. These facts were validated by subjecting the complexes to relative and absolute binding free energy methods of MMGB/PBSA and WaterSwap. A strong agreement on the system stability was disclosed that supported ligand high affinity potential for the receptors. Collectively, this work sought to provide preliminary experimental data of existing anti-viral drugs as a possible therapy for COVID-19 infections and a new peptide-based vaccine for protection against this pandemic virus.

Multiepitope Subunit Vaccine Design against COVID-19 Based on the Spike Protein of SARS-CoV-2: An In Silico Analysis.

The global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, has resulted in a negative impact on human health and on social and economic activities worldwide. Researchers around the globe need to design and develop successful therapeutics as well as vaccines against the novel COVID-19 disease. In the present study, we conducted comprehensive computer-assisted analysis on the spike glycoprotein of SARS-CoV-2 in order to design a safe and potent multiepitope vaccine. In silico epitope prioritization shortlisted six HLA I epitopes and six B-cell-derived HLA II epitopes. These high-ranked epitopes were all connected to each other via flexible GPGPG linkers, and at the N-terminus side, the sequence of Cholera Toxin ß subunit was attached via an EAAAK linker. Structural modeling of the vaccine was performed, and molecular docking analysis strongly suggested a positive association of a multiepitope vaccine with Toll-like Receptor 3. The structural investigations of the vaccine-TLR3 complex revealed the formation of fifteen interchain hydrogen bonds, thus validating its integrity and stability. Moreover, it was found that this interaction was thermodynamically feasible. In conclusion, our data supports the proposition that a multiepitope vaccine will provide protective immunity against COVID-19. However, further in vivo and in vitro experiments are needed to validate the immunogenicity and safety of the candidate vaccine.

Relationship of S/R warfarin ratio with CYP2C9 genotypes in Pakistani population.

Warfarin is administered as a racemic preparation of R- and S-enantiomers. S-warfarin is more potent than R-warfarin, so changes in blood levels of S-warfarin affect the anticoagulant response. This study was carried out to determine the effect of CYP2C9*2 and CYP2C9*3 polymorphisms on S/R warfarin ratio. A single blood sample was collected 12-16 hours after drug administration from 170 stable patients fulfilling the criteria. Genotyping of the CYP2C9 polymorphisms was done by polymerase chain reaction-restriction fragment length polymorphism assay. S- and R-warfarin enantiomers extraction from plasma was accomplished by a validated HPLC method. The concentration of S-warfarin was significantly different among CYP2C9 genotypes (p =0.018) whereas there was no effect on R-warfarin (p =0.134). There was statistically significant effect of different CYP2C9 genotypes on S/R warfarin ratio (p=0.000). It is concluded that CYP2C9 polymorphisms influence CYP2C9 enzymatic activity in turn affecting S-warfari levels but not R-warfarin, thus leading to different S/R warfarin enantiomers ratio among different CYP2C9 genotypes.

Evaluation of Three Rapid Screening Tests for Detection of Hepatitis C Antibodies on Mass Scale.

Hepatitis C virus (HCV) is a leading health problem across the globe. Only 20% of HCV positive individuals know their positive disease status. Effective HCV screening tests are required to screen both general and high-risk populations and identify the silent cases of HCV. In this study, we analyzed the performance of three rapid HCV screening kits. A total of 300 subjects from three populations groups, were enrolled from Rawalpindi and Islamabad cities of Pakistan. The three groups were blood donors (n = 50), pregnant women (n = 50), and hepatitis C positive individuals (200). Blood samples of all the individuals were screened on three rapid screening tests for anti-HCV: CTK Biotech's OnSite HCV Ab Rapid Test, SD Bioline One Step anti-HCV test, and Intec Products Advanced Quality Rapid Anti-HCV Test. The performance of these three rapid tests was also compared with the Roche Anti-HCV II test performed on the cobas 601 platform based on the electrochemiluminescence immunoassay principle. In total, 300 samples were analyzed in this study, out of which 208 were positive for anti-HCV positive and 92 were negative for anti-HCV. The sensitivities of the Intec product, SD Bioline, and CTK Biotech were 98.56%, 97.59%, and 95.67%, respectively. The specificity of SD Bioline and CTK Biotech were 100%, whereas Intec products showed 98.91% specificity. The positive predictive value (PPV) of SD Bioline and CTK Biotech was 100%, but Intec products showed 99.51% PPV. The negative predictive values of the Intec product, SD Bioline, and CTK Biotech were 96.80%, 94.84%, and 91.09%, respectively. There is a dire need to speed up HCV screening to achieve the targets in the World Health Organization global viral hepatitis strategy (2016-2021). The rapid tests evaluated in this study can be used in hepatitis screening on much larger scales.

The World after Ebola: An Overview of Ebola Complications, Vaccine Development, Lessons Learned, Financial Losses, and Disease Preparedness.

The 2014-2016 Ebola outbreak in West Africa was the largest of its kind with 11,000 deaths and approximately 28,637 affected cases. The aim of the study was to analyze the global situation after the Ebola outbreak including Ebola complications, vaccine development, lessons learned, financial losses, and disease preparedness. We searched in PubMed, Google, and Google Scholar by using keywords Ebola virus, Ebola vaccine development and Ebola virus transmission, the world after Ebola, financial losses by Ebola outbreak, and disease preparedness. Ebola virus disease is a complex disorder associated with gastrointestinal, hepatic, renal, respiratory, cardiovascular, and neurological complications. Ebola virus persisted in the semen of male infected patients for 470 to 565 days, and the chances of viral transmission by sexual contacts remained high even after patient recovery. There are several reports of extreme socioneuropsychological disorders in Ebola survivors and Ebola healthcare workers. There is no Food and Drug Administration-approved drug or vaccine for Ebola. Many research groups are working to develop a vaccine against Ebola by using different biotechnology techniques. Some vaccine candidates, including replicating vesicular stomatitis virus and Chimpanzee adenovirus-3, have entered phase III clinical trials and are expected to receive clinical licensing in coming years. The West African Ebola epidemic caused a financial loss of $6 billion in Africa and an additional global economic loss of more than $12 billion. After the Ebola epidemic, four global commissions were established for disease preparedness. A proposition was also forwarded to the World Health Organization for the establishment of the Centre for Emergency Preparedness and Response for the disease management. The devastating Ebola epidemic opened the window for disease preparedness to face any future disease epidemic.

Hepatitis elimination by 2030: Progress and challenges.

Globally, over 300 million people are living with viral hepatitis with approximately 1.3 million deaths per year. In 2016, World Health Assembly adopted the Global Health Sector Strategy on viral hepatitis to eliminate hepatitis by 2030. Different World Health Organization member countries are working on hepatitis control strategies to achieve hepatitis elimination. So far, only 12 countries are on track to achieve hepatitis elimination targets. The aim of the study was to give an update about the progress and challenges to achieving hepatitis elimination by 2030. According to the latest data, 87% of infants had received the three doses of hepatitis B virus (HBV) vaccination in the first year of their life and 46% of infants had received a timely birth dose of HBV vaccination. There is a strong need to improve blood and injection safety. Rates of hepatitis B and C diagnosis are very low and only 11% of hepatitis B and C cases are diagnosed. There is a dire need to speed up hepatitis diagnosis and find the missing millions of people living with viral hepatitis. Up to 2016, only 3 million hepatitis C cases have been treated. Pricing of hepatitis C virus drugs is also reduced in many countries. The major hurdle to achieve hepatitis elimination is lack of finances to support hepatitis programs. None of the major global donors are committed to invest in the fight against hepatitis. It will be very difficult for the low and middle-income countries to fund their hepatitis control program. Hepatitis elimination needs strong financial and political commitment, support from civil societies, and support from pharmaceutical and medical companies around the globe.

Frequency of Common VKORC1 Polymorphisms and Their Impact on Warfarin Dose Requirement in Pakistani Population.

Polymorphisms in vitamin K epoxide reductase complex subunit 1 (VKORC1) gene lead to interindividual variability in warfarin dose requirement. The characterization of genotype frequency distribution is required in different populations for construction of customized dosing algorithms to enhance the efficacy and reduce the toxicity of warfarin therapy. This study was carried out in Pakistani population to evaluate the contribution of common VKORC1 polymorphisms to warfarin therapy. A total of 550 stable patients taking warfarin were enrolled after medical history, physical examination, and laboratory investigations. Single blood sample was collected after informed consent. Genomic DNA was extracted and genotype analysis for VKORC1 1173C>T and VKORC1-1639G>A polymorphisms was done by polymerase chain reaction-restriction fragment length polymorphism assay. A number of samples were also analyzed by direct DNA sequencing for validation of results. Data were analyzed using SPSS version 20. Genotype frequency distributions of VKORC1 1173C>T and VKORC1-1639G>A were found to be different from other populations. Both of these polymorphisms did not demonstrate significant effect on warfarin dose requirement. Although Cytochrome P450 2C9 (CYP2C9) and VKORC1 polymorphisms together attributed only 3.8% variability in warfarin dose but it was statistically significant ( p value = .004). It is concluded that there is a need to study genotype frequency distribution and their effect on warfarin dose variability among different populations due to diversity in outcome. At the same time, no effect on warfarin dose variation explained by VKORC1 polymorphisms and small variability explained by studied genotypes stresses the need for exploration of more genetic and nongenetic factors in Pakistani population.

Prevalence of hepatitis C in people who inject drugs in the cities of Rawalpindi and Islamabad, Pakistan.

Pakistan has the second highest burden of hepatitis C (HCV) in the world. The major route of HCV transmission is contaminated blood or needle sharing. Seventy percent of people who inject drugs (PWIDs) shared needles at some time in their addiction history. The aim of the present study was to estimate the prevalence of HCV in PWIDs in cities of Pakistan. We enrolled 100 PWIDs from the Rawalpindi and Islamabad cities of Pakistan. Blood samples were taken in collection tubes and were subjected to HCV screening by using three rapid HCV screening kits including one step anti-HCV test, onsite HCV Ab rapid test and advance quality rapid anti-HCV test. All 100 blood samples were also subjected to HCV detection by using Elecsys anti-HCV II performed on the Roche Cobas 601 platform based on the ECLIA principle. Seventy-two percent of PWIDs showed the presence of HCV antibodies using the Roche anti-HCV II ECLIA test. We also compared the performance of different rapid kits in comparison with the anti-HCV II by Roche. The sensitivity of CTK kit was 84.72%, which was almost equal to the sensitivity by the SD Bioline HCV and Advanced Quality Rapid HCV tests, which was 83.33%. All three kits showed 100% specificity and positive predictive values. The results showed that the three market competitors of HCV rapid test showed almost equal results. The prevalence of HCV is very high in PWIDs in the capital twin cities of Pakistan. There is dire need to initiate the administration of a hepatitis test and treatment program for both high-risk and the general HCV-positive population. This is the optimal way to achieve HCV control targets established by the United Nations Sustainable Development Goals and Global Health Sector Strategy by WHO.

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.

Frequency of Common CYP2C9 Polymorphisms and Their Impact on Warfarin Dose Requirement in Pakistani Population.

Polymorphisms in cytochrome P450 (CYP) 2C9 (CYP2C9) gene result in interindividual variability in warfarin dose requirement. There is a need for characterization of genotype frequency distribution in different populations for construction of customized dosing algorithms to enhance the efficacy and reduce the toxicity of warfarin therapy. This study was carried out in Pakistani population to evaluate the contribution of common CYP2C9 polymorphisms to warfarin therapy. A total of 550 stable patients taking warfarin were enrolled after medical history, physical examination, and laboratory investigations. Single blood sample was collected after informed consent. Genomic DNA was extracted, and genotype analysis for CYP2C9*2 and CYP2C9*3 polymorphisms was done by polymerase chain reaction-restriction fragment length polymorphism assay. A number of samples were also analyzed by direct DNA sequencing for validation of the results. Data were analyzed using SPSS version 20. Genotype frequency distribution of CYP2C9*2 and CYP2C9*3 was found to be different from other populations. Of these 2 polymorphisms, CYP2C9*2 did not demonstrate significant effect on warfarin dose requirement, whereas CYP2C9*3 did show significant effect ( P value = .012). It is concluded that there is a need to study genotype frequency distribution and their effect on warfarin dose variability among different populations due to diversity in outcome.

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 12.5 ng/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.

EFFECT OF NEBIVOLOL ON TONE OF TRACHEAL MUSCLE OF GUINEA PIG.

BACKGROUND: The use of ß-blockers is limited by adverse effects such as bronchospasm in asthmatics. Third generation ß-blockers such as nebivolol may show better tolerability in asthmatic subjects because they lack ß-blocker induced bronchoconstriction. Methods: Effects of nebivolol on the tracheal muscle strips prepared from ovalbumin-sensitised guinea pigs of both sexes were studied. Two sets of experiments were designed after dividing the animals randomly into two groups. Using oxygenated Krebs-Henseleit solution as the nutrient medium, the trachealis muscle activity was measured with isometric force displacement transducer and recorded on 4-channel Oscillograph. RESULT: Nebivolol 10(-6) M did not produce significant effect on contractions evoked by histamine in concentrations ranging from 10(-7) M to 10(-3) M. The mean of amplitude of contraction for different concentrations of histamine were calculated and compared with the group treated with histamine only. Mean of amplitude of contraction, percent responses and percent deviations when compared with the control group were insignificant (p>0.05). CONCLUSION: Nebivolol did not affect the tone of airway smooth muscle in ovalbumin-sensitised guinea pigs. Nebivolol may be considered safe in patients with airway disease however, further clinical evaluation and exploratory work is required.

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-oo), Cmax, Tmax, t½, 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-oo) and Cmax 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.

Pharmacokinetics of standard dose regimens of rifampicin in patients with pulmonary tuberculosis in Pakistan.

BACKGROUND: This clinical study investigated the pharmacokinetics of standard doses of rifampicin (RMP; 450 and 600 mg) in pulmonary tuberculosis (TB) patients in the context of its high sterilizing potential and the increased frequency of multidrug-resistant TB in Pakistan. The objective of this study was to determine the sufficiency or inadequacy of peak plasma levels of RMP in pulmonary TB patients after the administration of standard doses. METHODS: Twenty adult patients with newly diagnosed pulmonary TB consented to participate in the study. Blood sampling for pharmacokinetic assessment of RMP was done after at least 14 days of regular daily treatment to ensure steady state. Plasma concentrations of RMP were determined by a validated high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection. RESULTS: The peak plasma concentration of RMP at 2 h was subtherapeutic, i.e. 3.76 ± 1.23 mg/l (range 1.80-6.62), in all of our patients. Out of 20 patients, 13 (65%), had 2-h plasma concentrations below 4 mg/l. CONCLUSIONS: This study reports evidence of suboptimal RMP concentrations in a small group of Pakistani TB patients and highlights the need for larger clinical studies to identify possible reasons and consequences of low RMP levels in terms of treatment outcomes. Quality control problems in local drug preparations used widely among TB patients in Pakistan need to be addressed as a matter of urgency.

Determination of pharmacokinetics of flurbiprofen in Pakistani population using modified HPLC method.

Pharmacokinetics of flurbiprofen has been studied in different populations, especially in Caucasian. However, there are very few studies reported from Eastern part of world. Previous studies suggested that genetic and environmental factors may cause inter-individual differences in flurbiprofen disposition, so we investigated the pharmacokinetics of flurbiprofen in Pakistani subjects. A single oral dose of 100 mg of flurbiprofen was administered to 22 healthy male Pakistani adults after overnight fasting for 10 h. Periodical blood sampling was done at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, and 24 h after dosing. Plasma concentration of flurbiprofen was determined by a modified high-performance liquid chromatography method, which was simple, sensitive, less time consuming and economical with ordinary internal standard. The method was validated according to ICH guidelines and was found to be sensitive, accurate and precise. The pharmacokinetic parameters observed in Pakistani subjects when compared with other populations (USA, UK, Canadian, French, and Indian) did not show considerable ethnic differences. However, one subject's data was suggestive of being poor metabolizer of flurbiprofen which supports the presence of CYP2C9 polymorphism contributing to inter-individual differences in flurbiprofen disposition. Pharmacogenomic studies are needed to verify this hypothesis.

Pharmacological basis for use of Pistacia integerrima leaves in hyperuricemia and gout.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Pistacia integerrima Stew ex. Brandis is an important component of commonly dispensed traditional dosage forms. We wished to determine whether polyphenolic constituents of this plant could be useful in oxidative stress and have potential to counter hyperuricemia. MATERIAL AND METHODS: Radical scavenging activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and xanthine oxidase (XO) inhibitory activity assay in vitro. Fructose (FRS) induced hyperuricemic animal model was used to asses the serum uric acid (UA) lowering effect by plant products. RESULTS: Ethyl acetate and n-BuOH fractions had the highest DPPH radical scavenging activity. Fifty percent inhibitory concentration (IC(50)) was 6 and 7.6 microg/ml respectively. It was less than quercetin (IC(50) 0.95 microg/ml) and ascorbic acid (IC(50) 1.76 microg/ml). Xanthine oxidase inhibitory activity was comparable between n-BuOH and EtOAc (IC(50) 19 and 20 microg/ml) extracts but less than quercetin (IC(50) 0.65 microg/ml) and allopurinol (IC(50) 0.10 microg/ml). The antioxidant activity as well as the inhibitory activity towards the enzyme XO by quercetin-3-O-beta-d-glucopyranoside (5), kaempferol-3-O-beta-d-glucopyranoside (6), quercetin-3-O-(6''-O-syringyl)-beta-d-glucopyranoside (7), kaempferol-3-O-(4''-O-galloyl)-alpha-l-arabinopyranoside (8), rutin (4) together with aglycons, quercetin (1), kaempferol (2) and apigenin (3) was promising to continue in vivo hypouricemic studies. Ethyl acetate extract had dose dependent UA lowering effect in hyperuricemic mice. This effect was comparable with quercetin but less than allopurinol. CONCLUSIONS: These findings are encouraging to plan clinical studies in hyperuricemic patients.