Adenovirus–MVA heterologous prime–boost using a PfMSP1 antigen in

Adenovirus–MVA heterologous prime–boost using a PfMSP1 antigen insert is a leading viral vectored regime for antibody and T cell induction against this blood-stage P. falciparum antigen [3] and [5]. As a protein-adjuvant comparator, we used a Pichia pastoris-expressed recombinant PfMSP119 [33], adjuvanted by Montanide ISA720 (Seppic, France). Montanide

ISA720 is a squalene-based water-in-oil emulsion which has been shown to be a potent adjuvant in both animal and human studies [34], [35], [36] and [37]. Here we describe and compare in detail the immunogenicity of PfMSP1 Bortezomib supplier vaccines using a novel combination of three subunit vaccine platforms: simian adenovirus AdCh63 [5] and [38]; MVA; and recombinant protein in Montanide ISA720. We report that, when combined, these technologies can achieve simultaneous antibody and T cell responses

which DAPT ic50 equal, or in some cases surpass, the best immune responses achieved with either technology alone. We describe in detail the responses induced, with data on antibody isotypes and avidity, splenic antibody secreting cell counts, T cell quality, and response longevity. All procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act Project Licence and were approved by the University of Oxford Animal Care and Ethical Review Committee. 5–6 weeks old female BALB/c (H-2d) and C57BL/6 (H-2b) mice (Harlan Laboratories, Mephenoxalone Oxfordshire, UK) were anesthetized before immunization with medetomidine (Domitor, Pfizer) and ketamine (Ketaset, Fort Dodge) and revived subsequently with Antisedan reversal agent (Pfizer). All immunizations were administered intramuscularly (i.m.) unless otherwise specified, with vaccine divided equally into each musculus tibialis. The creation of simian adenovirus 63 (AdCh63) and modified vaccinia virus Ankara (MVA) vectors encoding the PfM128 antigen is described elsewhere [5]. Briefly,

this antigen is a bi-allelic fusion incorporating the MSP142 antigen from the K1/Wellcome and 3D7/MAD20 P. falciparum strains fused in tandem alongside four blocks of conserved sequence from the remainder of the 3D7 strain MSP1 molecule (blocks 1, 3, 5 and 12). The MVA used in the current study differs from the previously published vector [3] in that it lacked the green fluorescent protein (GFP) marker. To generate the markerless MVA expressing PfM128, the antigen was cloned into a transient-dominant shuttle vector plasmid such that PfM128 was expressed from the vaccinia P7.5 promoter, and inserted into the TK locus of MVA. The plasmid also expresses a GFP marker [39]. This plasmid was transfected into chicken embryo fibroblast cells (CEFs) infected with MVA expressing red fluorescent protein (RFP), as previously described [3]. Recombinant MVAs were generated by homologous recombination between regions of homology at the TK locus of MVA and in the plasmid shuttle vector.

He was one of the first physicians to attain formal “Med-Peds” tr

He was one of the first physicians to attain formal “Med-Peds” training, completing a Pediatric

residency at Cornell after an Internal Medicine internship at Johns Hopkins. Karzon’s basic research career began with a fellowship to study Newcastle disease virus, and continued during his first faculty appointment at the University of New York in Buffalo (1952–1968), where he began scientific investigations into polio, measles, canine distemper, rhinderpest, mumps, rubella, echovirus, and influenza. Going back to his childhood, he also discovered and conducted studies on viruses from amphibians and reptiles. In 1968 Karzon accepted an appointment as Chairman of Pediatrics at Vanderbilt University School of Medicine. There he continued to promote work on infectious diseases, and through skilful recruitment and development of local talent helped build Cyclopamine a strong Raf inhibitor program devoted to the study of basic microbial pathogenesis and clinical research focused on vaccine evaluation. Later in his career as he stepped away from the administrative duties of Chairman (1986), he focused his accumulated wisdom on HIV vaccine development efforts and on basic studies of respiratory syncytial virus, which have been the areas of major focus in our own scientific careers. He was an important figure in guiding many young investigators as they established careers in academic medicine

and developed strategies for asking research questions. Critical thinking was serious business for Karzon, and he was prepared with a full cup of sharpened #2 pencils to extensively

comment and query the documents presented to him by his protégés. Throughout his professional life, Karzon remained profoundly influenced by the children with polio whom he had encountered at the Sydenham Hospital. They not only shaped his research interests, but also motivated his advocacy for children in his academic and administrative work, his community activities, and his consultative efforts involving vaccine policy and regulation. Following the Phosphoprotein phosphatase success of the polio vaccine campaign in the 1950s and early 1960s, he carried that momentum and energy into building a medical infrastructure to provide care to all children. When he arrived in Nashville, the community considered the Junior League Home for Crippled Children as the primary site for compassionate caring of sick children. The Junior League of Nashville had originally built the Home for Crippled Children in the early 1900s to focus on the convalescent care of indigent victims of polio. As polio receded in the 1950s, the Junior League Home for Crippled Children merged with the Nashville Chapter of the National Council for Jewish Women’s Convalescent Home for children with noninfectious diseases, and with the support of the Al Menah Shriners and both private and academic physicians, the Home for Crippled Children began to address the broader spectrum of health care needs specific to children.

It is likely that his lasting legacy will be the decision to intr

It is likely that his lasting legacy will be the decision to introduce the new name for the journal in a bid to allow it to take its rightful place in the range of international publication options for physiotherapists. Professor Hodges has served as a figurehead for the journal both nationally and internationally, and will be missed. His departure

is compulsory as he has served this website the maximum number of terms provided for by the Australian Physiotherapy Association. Associate Professor Ada was appointed Scientific Editor in June 2005 and will remain as a member of the Editorial Board in an honorary capacity during 2010 to ensure a smooth transition. During her time at the helm she revised and expanded the Author Guidelines to provide models for submission of a number of types of paper. She introduced structured headings for papers and devised downloadable Templates for the submission find more of Tables and Figures. Many submitting authors have commented positively on the assistance the Guidelines provide. She edited papers extensively so that they are consistent in terminology and very readable. When Associate Professor Ada became Scientific

Editor, the 2004 Impact Factor was 1.021; she leaves the journal with the 2008 impact factor at 1.948. Every year has shown growth under her Editorial guidance. In 2005 the journal received 82 submission; in 2009 there were 105, all of which Associate Professor Ada managed through the review process. The workload on this aspect of the journal alone increased by 25%. It is also timely to acknowledge the contributions to the Dipeptidyl peptidase Editorial Board of Associate Professor Linda Denehy who completed her term of office in December, and Associate Professor Sandy Brauer who has been re-appointed for a further term. Other changes include the appointment of Associate Professor Lisa Harvey, Dr Julia Hush, and Dr Terry Haines to the Editorial Board. Members continuing on the Editorial Board are Associate Professor Michelle

Sterling and Professor Nicholas Taylor. The Editorial Board is grateful for the substantial contribution of these dedicated and skilled individuals. Under the combined stewardship of Professor Hodges and Associate Professor Ada, AJP has grown and matured as a general journal of physiotherapy. We look forward to the continued growth and international positioning of the newly named Journal of Physiotherapy. “
“Physiotherapists commonly assess and treat upper extremity disorders. Passive joint mobilisation or manipulation has been shown to be effective in disorders such as adhesive shoulder capsulitis, non-specific shoulder pain or dysfunction (Ho et al 2009), shoulder impingement syndrome (Kromer et al 2009), lateral epicondylalgia (Bisset et al 2005), and carpal tunnel syndrome (O’Connor et al 2003). Measurement of passive movement is indicated in order to assess joint restrictions and to help diagnose these disorders.

O/IND/R2/75 vaccine strain was received from the virus seed labor

O/IND/R2/75 vaccine strain was received from the virus seed laboratory, IIL, Hyderabad. O/HAS/34/05 virus was used for experimental infection of buffalo. O/HAS/34/05 virus is homologous to O/IND/R2/75 (r1 value > 1.00) [11]; and was

isolated from epithelial tissue of a suspected FMD case in a non-vaccinated buffalo from Sirsa District, Haryana Metformin State. Challenge virus O/HAS/34/05 was prepared by passaging in the tongues of buffalo calves as described for cattle by Nagendrakumar et al. [12]. Briefly, one buffalo calf was inoculated intradermolingually with BHK 21 monolayer adapted O/HAS/34/05 virus (105 TCID50). The tongue epithelium was collected 48 h post inoculation. For a second passage, epithelial tissue was collected from vesicles PLX4032 research buy and after trituration in 0.04 M phosphate buffer followed by centrifugation at 3000 rpm; the clear supernatant was used

to inoculate (intradermolingually) the 2nd buffalo. The same procedure was followed for third buffalo passage. Then the tongue epithelium was collected from third passage buffalo and 20% W/V virus suspension was prepared. To make the glycerol stock 50% of sterile glycerol was added to the virus suspension and stored at −20 °C. The virus was then titrated in buffalo calves to establish the buffalo infective dose 50 values (BID50). Murrah male buffalo calves (n = 24; 6–12 months of age) and crossbred male cattle calves (n = 12; 6–12 months of age) were obtained from the holding farm tuclazepam of IIL, Hyderabad. These animals were reared in the farm from one month of age and were screened by 3 rounds of testing for FMDV-non-structural protein (NSP) antibodies using PrioCHECK® FMDV NS kit (Prionics Lelystad B.V., The Netherlands)

and structural antibodies [13]. All the animals were negative against both NSP and structural antibodies in all the three rounds of testing. In addition, the animals were tested for the absence of virus in the oesophago-pharyngeal fluids (Probang samples) by inoculation of primary bovine thyroid cells [14] followed by antigen ELISA [15] and RT-PCR [16]. Monovalent FMD vaccine incorporating O/IND/R2/75 (7 μg/dose) FMDV inactivated antigen was formulated with Montanide ISA 206 (Seppic, France) as a water-in-oil-in-water (W/O/W) emulsion. One group of buffalo calves (GrI; n = 6) and a second group of cattle calves (GrII; n = 6) were administered with 2.0 ml of formulated vaccine by intra-muscular route whereas a third and a fourth group of buffalo (GrIII; n = 6) and cattle (GrIV; n = 6) calves remained unvaccinated. Donor buffalo (n = 12) were inoculated with 105 BID50 of buffalo passaged O/HAS/34/05 FMDV by the intradermolingual route at 24 h before contact challenge.

Tivendra Kumar, Centre for Health Research and Development, Socie

Tivendra Kumar, Centre for Health Research and Development, Society for Applied Studies, Delhi. Vinohar Balraj, Professor of Community Health, Christian Medical College, Vellore. Jayaprakash Muliyil, Academic Officer, Christian Medical College, Vellore. Gagandeep Kang, The Wellcome Trust Research Laboratory, Christian Medical Medical College, Vellore. Jacob John, Associate Professor of Community Health, Christian Medical College, Vellore. Mohan V. Raghava, Associate Professor of Community Health, Christian Medical College, Vellore. Rajiv Sarkar, Department of Gastrointestinal Sciences, Christian Medical College, Vellore.

Umesh D. Parashar, Head, Viral Vorinostat Gastroenteritis Section, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta. Nicholas C. Grassly, Professor of Infectious Disease & Vaccine Epidemiology, Imperial College, London. Mathuram Santosham, Professor of International Health and Pediatrics, Johns Hopkins Bloomberg, School of Public Health, Baltimore.

“The World Health Organization (WHO) has recommended oral rotavirus vaccines for all infants worldwide [1]. As of May 20, this website 2014, 60 countries worldwide and 26 GAVI-eligible countries had introduced rotavirus vaccine (RV) into their national immunization programs [2]. (Fig. 1) Major barriers to more rapid introduction of rotavirus vaccines in low-resource settings have been related to vaccine cold chain constraints in some countries and limited product-of-choice availability for others. Thus, the availability of additional, affordable rotavirus vaccines is a high priority to enhance rotavirus STK38 disease control efforts. Clinical trials under real-world conditions in low-resource countries established the public health benefit

of RotaTeq® (Merck & Co.) and Rotarix® (GlaxoSmithKline), and informed the WHO recommendation for their use [1], [3], [4] and [5]. Much has been written about the lower point estimates of efficacy in these trials compared with trials performed in higher resource settings. Among the reasons given for the lower efficacy are higher maternal antibody in low-resource settings, environmental enteropathy, differences in the gut microbiome among children in different resource settings, nutritional status, breastfeeding practices and interference by oral poliovirus vaccines [6], [7], [8] and [9]. In addition to these factors, we propose that the contribution of study design differences should be considered when comparing point estimates of efficacy across trials. In addition, the biologic factors and study design factors may be interrelated; for example, the higher antibody in low resource settings may be due to both an increased exposure to rotavirus and to the younger age at administration of routine childhood vaccines, including rotavirus vaccines.

Lancefield and Hare subsequently identified GBS in vaginal swabs

Lancefield and Hare subsequently identified GBS in vaginal swabs in 1935 [2] and in 1938 Fry described three fatal cases in post-partum women [3]. Reports of neonatal disease from GBS were sporadic until the early 1960s when GBS became recognized as a leading cause of early neonatal sepsis in the USA [4]. By the 1970s it had become the dominant pathogen in the early neonatal period [5]. By the early 1980s GBS had become the most common cause of neonatal sepsis and meningitis in a number of developed countries [6], [7] and [8]. In the past five years, GSK-3 assay late-onset (LO) GBS disease has been associated with case reports of transmission via infected breast milk [9]

raising questions about mode of acquisition and transmission of this enteric pathogen and the development of neonatal disease. Although GBS is not just a neonatal disease, the disease incidence and severity is highest during the first 90 days of life. Early onset (EO) GBS disease (disease presenting in the first six days of life) accounts for approximately 60–70% of all GBS disease. GBS serotypes Ia, Ib, II, III

and V are responsible for most EO disease [10] and [11]. In contrast, serotype III predominates in LO disease, which may be acquired perinatally, click here nosocomially or from the community. [12] In the USA EO disease rates have declined from 1.4 per 1000 live births in 1990 [13] to at 0.28 per 1000 live births in 2012 [14] mainly attributed to the implementation of universal screening for GBS rectovaginal colonization in pregnant women and intrapartum antibiotic prophylaxis. However, the incidence of LO disease has remained static at between 0.3 and 0.4 per 1000 births

since 1990 [14]. This amounts to 28,100 cases and 1865 deaths annually in the USA [14]. Although the epidemiology of GBS in resource-rich countries is well documented, its contribution to the burden of neonatal infection in low/middle income countries has proved more difficult to assess. GBS has been reported as the predominant cause of neonatal sepsis in South Africa and Kenya [15], [16] and [17] as well as an important cause of meningitis in Malawi and and Kenya, but Asian studies have reported a much lower incidence [18], [19] and [20]. A recent systematic review reported that the overall incidence of GBS in resource-poor settings ranged between 0 and 3.06 per 1000 live births [21]. GBS colonizes the rectum and vagina, and maternal colonization is a pre-requisite for EO disease and a risk factor for LO [22] and [23]. In resource-rich countries an estimated 20–30% of pregnant women are colonized with GBS [23] and [24], approximately 50% of their babies become colonized and 1% progress to develop invasive disease. EO disease may occur rapidly; signs of sepsis are evident at birth or within 12 h in over 90% of cases (98% within the first 12 h) [12].

(2008) who hypothesised: ‘[t]hat by exploring differences between

(2008) who hypothesised: ‘[t]hat by exploring differences between schools, we may be able to determine school factors that are, for better or worse, having an impact on children’s risks of obesity.

At the same time, we may be able to highlight ‘hot’ and ‘cold’ spots of obesity so allowing better targeting of resources to those communities in greatest need. To test this hypothesis Procter et al. (2008) employed a ‘value-added’ MK-8776 technique similar to those developed in economics and regularly used to assess the educational impact of schools (Amrein-Beardsley, 2008 and Rutter, 1979). In education, an individual’s value-added score is the change in outcome (e.g. test score) during the period of their schooling. In order to compare school performance the individual scores are aggregated, and it becomes necessary to adjust for differences in school composition which could bias the scores (Amrein-Beardsley, 2008 and Rutter, 1979). Procter et al. (2008) accounted for the ethnic and socioeconomic composition of 35 primary schools in Leeds, England, who were participating in the Trends study to rank schools according to their mean observed and expected residual pupil weight status and ‘value-added’ score. The authors found that there was little

similarity between the ‘value-added’ and expected residual BIBW2992 datasheet rankings and concluded that this lent credence to the hypothesis that differing school environments have differential impacts upon their PDK4 pupils (Procter et al., 2008). As a result they suggested that obesity prevention efforts be targeted rather than

population wide as ‘hot’ and ‘cold’ schools for obesity had been identifiable, and hence future research should focus on such schools. Acknowledging the fallibility of such ‘league tables’, Procter et al. (2008) also suggested that these analyses should be replicated across a number of years to test the validity of the findings (Goldstein and Spiegelhalter, 1996). This study evaluates and expands upon the technique proposed by Procter et al. (2008) using repeated cross-sectional data from a large routine data source (the National Child Measurement Programme (NCMP)) to examine the potential differential impact of primary schools on children’s weight status. The English NCMP was introduced in 2005 to monitor progress towards a public service agreement to reduce the prevalence of obese primary school aged children (Dinsdale and Rutter, 2008 and South East England Public Health Observatory, 2005). Unless individuals or schools are actively opted out, all Reception (4–5 year olds) and Year 6 (10–11 year olds) pupils in state maintained primary schools have their height and weight measured by a health professional (Dinsdale and Rutter, 2008). Five years of NCMP data (2006/07–2010/11, involving 57,976 pupils) from Devon local authority were used in this study.

Conversely, an increased sICAM release was observed for H441 in M

Conversely, an increased sICAM release was observed for H441 in MC, whereas no sICAM response was detectable for H441 in CC. This might

be due to a higher differentiation and polarisation of the H441 considering a well-developed apical membrane with microvilli concluding an altered shedding of adhesion molecules. Furthermore, an increased uptake (compared NVP-AUY922 mouse to a concentration of 60 μg/ml, as used for the transport experiments) was observed for the direct exposed H441 but not in the ISO-HAS-1 on the bottom side in which no fluorescence signals of NPs could be detected. These findings corroborate the above mentioned conclusion. These results also corroborate the observation by Kasper et al. [9], which described cross-talk between direct aSNP-exposed H441 with ISO-HAS-1 resulting in an inflammatory response of the endothelial Selleck 3-MA layer, which did not have a direct contact to NPs. A reason for the endothelial sICAM release may also be due to the elevated LDH release of the H441 and reduced TER. These finding could be attributed to the presence of necrotic cells at these very high concentrations. LDH, ATP and other

cytosolic components, which are released by necrotic cells, are known to cause inflammation. The induction of inflammatory processes induced by cell damage play also a significant role in the development of acute lung injury (ALI) or obstructive lung diseases (COPD). High concentrations such as 300 μg/ml used in this study probably exceed concentrations of NPs which may occur during inhalation processes in vivo, but they serve very well as a positive control for the in vitro setting. In consequence, subsequent approaches would have to take into

account effects caused by long-term or repeated exposure to nanoparticle in lower doses as it may occur in the development of obstructive lung diseases. According to this study, flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. Furthermore, the coculture model H441/ISO-HAS-1 represents a suitable model to study nanoparticle interactions with the alveolar epithelial barrier in vitro. It Montelukast Sodium allows an investigation into cellular uptake/transport of nanoparticles as well as cell–cell communication processes after nanoparticle exposure at the alveolar-capillary site. In addition to an induction and release of inflammatory signals after NP exposure, which causes local effects on cells of the alveolar barrier, this study proposes forwarded inflammatory signals which may provoke further systemic effects. We are currently investigating a primary cell coculture model of the alveolar-capillary barrier consisting of primary human ATII (alveolar type II cells) and HPMEC (human pulmonary microvascular endothelial cells) to compare these cells to the model described in these studies.

While the extent of immune enhancement

While the extent of immune enhancement VE 821 of susceptibility/infectiousness by different infection sequences has been more difficult to estimate, there is some evidence to suggest that it might also vary between serotypes [14]. Furthermore, recent work suggests that such immune enhancement is important for serotype persistence in the presence of transmission heterogeneity [20]. The potential impact of vaccination on dengue transmission dynamics in Thailand and Vietnam has been explored in two recent publications by Chao et al. [21] and Coudeville et al. [22] using an agent-based model and an age-specific compartmental model, respectively. Both of these studies found that

vaccines with efficacy of 70–90% against all serotypes have the potential to significantly reduce the frequency and magnitude of epidemics on a short to medium term. However, while both of these models do account

for some sources of heterogeneity between serotypes, for example, differences between the serotypes in transmission intensity, they do not systematically examine the potential impact of these heterogeneities in the context of partially effective vaccines. Here, we use an age-stratified dengue transmission model to assess the potential impact of vaccines with high efficacy against dengue serotypes 1, 3 and 4 and low efficacy against dengue serotype 2 in a hyperendemic Thai population. We explore multiple disease/transmission scenarios to identify those that might lead to increases in clinically apparent cases and to identify the potential reductions in disease. Crucially, we evaluate the effects that certain serotype Trichostatin A manufacturer heterogeneities may have in the presence of mass-vaccination campaigns. We also explore overall, direct and indirect effects of reducing (or in some cases increasing)

infection and disease in vaccinated individuals vs. reductions in transmission population wide. We formulated a deterministic, age-stratified compartmental dengue transmission model that includes explicit vector dynamics as well as cross-protection and infectiousness enhancement between dengue serotypes. Humans are assumed to be born susceptible and can undergo up to two infections by heterologous serotypes. Mosquito vectors are classified whatever as susceptible or infected by each of the circulating serotypes. We focus on the dengue vaccine being developed by Sanofi-Pasteur that requires three doses to achieve high protection. Vaccination reduces the susceptibility of vaccinated humans to dengue infection. We also allow for immune mediated vaccine induced enhancement in transmissibility. Since the main objective of our study was to explore changes in the number of clinically apparent dengue cases, upon mass-vaccination, we made assumptions about the probability of developing clinically apparent disease following infection. These assumptions also allowed us to calibrate our model with data from surveillance systems.

Stock solution stability was proved for 9 days and evaluated Sta

Stock solution stability was proved for 9 days and evaluated. Stability of the drug in plasma samples was proved at LQC, HQC levels using six replicates each with its freshly prepared samples of same concentration. Reinjection reproducibility stability, benchtop stability, autosampler stability, freeze–thaw stability and long term stability was proved for drug in plasma samples. The reinjection

reproducibility was evaluated by comparing the extracted plasma samples that were injected immediately (time 0 h), with the samples that were re-injected after storing in the Vorinostat mouse autosampler at 4 °C for 26 h. Stability samples were kept on bench (Benchtop stability) for 25 h and processed along with freshly prepared standards and proved the stability for 25 h. The stability of spiked human plasma samples prepared and stored at 4 °C in autosampler (autosampler stability) was evaluated for 79 h. Freeze–thaw stability at −30 °C at 4th cycle was performed and proved for 3 cycles by comparing with freshly prepared samples. Long term stability was proved for 34 days with its freshly prepared standards at respective concentrations. All these stability samples % Accuracy was less than 15%. The stability was proved as per USFDA guidelines.13 The bioanalytical method described above was applied to determine acamprosate concentrations in plasma following oral administration Enzalutamide purchase of healthy human volunteers. These volunteers were contracted in APL Research

centre, Hyderabad, India and to each one of the 14 healthy volunteers were administered

a 333 mg dose (one 333 mg tablet) via oral with 240 ml of drinking water. The reference product CAMPRAL® tablets, Manufactured by Forest pharmaceuticals, INC. USA. 333 mg, and test product Acamprosate tablet (test tablet) 333 mg were used. Study protocol was approved by IEC (Institutional Ethical committee) and by DCGI (Drug Control General of India). Blood samples were collected as pre-dose (0) hr 5 min prior to dosing followed by further samples at 0, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.75, 6.5, 7.25, 8, 9.5, 12, 14, 18, 24, 30, 36, 48, 56, 60, 72, 84 and 96 h. After dosing, 5 ml blood sample was collected each pre-established time in vacutainers containing K2EDTA. A total of 50 (25 time points for reference, and 25 for test) time points were collected and centrifuged at 3200 rpm, 10 °C, 10 min. Then they were kept frozen at −30 °C until sample analysis. STK38 Test and reference were administered to same human volunteers under fasting conditions separately and these volunteers were washed minimum 9 days intervals as per protocol approved by IEC. Pharmacokinetics parameters from human plasma samples were calculated by a non-compartmental statistics model using WinNon-Lin5.0 software (Pharsight, USA). Blood samples were taken for a period of 3–5 times the terminal elimination half-life (t1/2) and it was considered as the area under the concentration time curve (AUC) ratio higher than 80% as per the FDA guidelines.