Healthy Adult Human Volunteers Research Articles

Plant formulation ATRICOV 452 in improving the level of COVID-19 specific inflammatory markers in patients

ImportanceDue to the huge demand for healthcare facilities, there is a need for safe therapeutic intervention which can reduce the need for extensive healthcare support. ObjectiveIn phase 1, to determine the safety of plant formulation in healthy volunteers and in phase 2, to validate its potential to improve the level of COVID-19 specific inflammatory markers. DesignSetting, and Participants: The phase 1 single fasting dose study was conducted on 24 healthy adult human volunteers to evaluate the safety and tolerability of plant formulation. The usage of plant formulation in humans was found safe and encouraged conducting Phase 2 clinical trial to evaluate the efficacy in 100 COVID-19 patients along with the standard of care. InterventionsPhase 1: plant formulation capsule of 500 mg single dose; Phase 2: plant formulation 1 gm thrice a day for 14 days. ResultsDuring the Phase 1 trial no adverse event was observed and all organ systems were normal in function. During the Phase 2 trial, 100 patients underwent randomization, 50 were assigned to receive plant formulation, and 50 to receive placebo. Three patients in the placebo and two patients in the plant formulation group had dropped out of the study. Hence, the primary analysis population included 95 patients (48 allocated to plant formulation and 47 to placebo). The COVID-19-specific inflammatory markers improved faster and became normal in the plant formulation treatment group. No one needed hospital care or oxygen. Conclusionsand Relevance: The investigational product, plant formulation (ATRICOV 452) has been found to be safe in phases 1 and 2. Further, in the phase 2 trial, ATRICOV 452 was effective at the dose of 1 mg, three times a day dose frequency to improve the level of COVID-19 specific inflammatory markers. Trial registrationPhase 1: CTRI Registration number: CTRI 2020/09/027660 Phase 2: CTRI Registration number: CTRI/2021/01/030795.

Checklist to select contract Research Organization for early phase Bioavailability/Bioequivalence Clinical Studies in Healthy Adult Human Volunteers

Nowadays health agencies of regulated markets are becoming stringent regarding the bio-studies. So clinical/contract research organization (CRO) need to be selected carefully after their detailed assessment. As per requirement of bio-study CRO should be assessed with the study specific checklist of questions before awarding the bio-study. Questions related to various services of study like: Clinic phase, analytical phase, Pharmacokinetic and statistical phase, ethics approval, QA/QC, record handing etc. and related to CRO capabilities are discussed here with their relevance to conclude the abilities of CRO for successful execution of bio-study. Hence, this paper focuses all possible questions which need to be assessed before selection of CRO mainly for Bioavailability/Bioequivalence (BABE) study in healthy adult volunteers. Properly selected CRO will help for smooth execution of study and quality report and subsequently, hassle-free approval of dossier submitted to regulatory agency.

Optimized copolymeric microstructured platforms for smart controlled delivery of an anticoagulant drug: Preparation, in vitro assessment and crossover study in healthy adult human volunteers

In the present study, novel micro-structured copolymeric carriers were developed based on the grafting technology where acrylamide was chemically crosslinked with different types of Eudragits® (NE30D, L100, RL30D, or RS30D) based on a 41*21 factorial design. The designed systems efficiently engulfed the anticoagulant drug dipyridamole (DIP), within their formed entangled mesh of crosslinked polymeric network. An optimized formulation, ECOP4 with a desirability-value of 0.706, (in which DIP is engulfed within a copolymeric network of acrylamide and Eudragit® RS30D) showed high engulfment capacity (97.13 ± 1.34%) and controlled DIP release over 8 h. FTIR studies revealed absence of interactions between DIP and the formed copolymer. ECOP4 was further inserted within an easily-administered safe raft forming system composed of a mixture of LM-pectin and gellan gum. A pharmacokinetic study was performed using human volunteers to determine DIP concentration in their plasma after administering the designed formulation using the high-performance liquid chromatography (HPLC) method. A crossover design was adopted comparing the designed formulation with Persantin® 25 mg tablets as a reference standard. Superior results were obtained for the optimized formulation regarding the measured pharmacokinetic parameters (AUC0-24h, Cmax, and Tmax) with a 2.31 fold increase in relative bioavailability, which reveals the usefulness of the designed grafted dipyridamole formulation in site-specific delivery system.

Discovery of Sisunatovir (RV521), an Inhibitor of Respiratory Syncytial Virus Fusion.

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo.

Wide-vergence, multi-spectral adaptive optics scanning laser ophthalmoscope with diffraction-limited illumination and collection.

Visualizing and assessing the function of microscopic retinal structures in the human eye is a challenging task that has been greatly facilitated by ophthalmic adaptive optics (AO). Yet, as AO imaging systems advance in functionality by employing multiple spectral channels and larger vergence ranges, achieving optimal resolution and signal-to-noise ratios (SNR) becomes difficult and is often compromised. While current-generation AO retinal imaging systems have demonstrated excellent, near diffraction-limited imaging performance over wide vergence and spectral ranges, a full theoretical and experimental analysis of an AOSLO that includes both the light delivery and collection optics has not been done, and neither has the effects of extending wavefront correction from one wavelength to imaging performance in different spectral channels. Here, we report a methodology and system design for simultaneously achieving diffraction-limited performance in both the illumination and collection paths for a wide-vergence, multi-spectral AO scanning laser ophthalmoscope (SLO) over a 1.2 diopter vergence range while correcting the wavefront in a separate wavelength. To validate the design, an AOSLO was constructed to have three imaging channels spanning different wavelength ranges (543 ± 11 nm, 680 ± 11 nm, and 840 ± 6 nm, respectively) and one near-infrared wavefront sensing channel (940 ± 5 nm). The AOSLO optics and their alignment were determined via simulations in optical and optomechanical design software and then experimentally verified by measuring the AOSLO's illumination and collection point spread functions (PSF) for each channel using a phase retrieval technique. The collection efficiency was then measured for each channel as a function of confocal pinhole size when imaging a model eye achieving near-theoretical performance. Imaging results from healthy human adult volunteers demonstrate the system's ability to resolve the foveal cone mosaic in all three imaging channels despite a wide spectral separation between the wavefront sensing and imaging channels.

Dynamic Threat Processing.

During real-life situations, multiple factors interact dynamically to determine threat level. In the current fMRI study involving healthy adult human volunteers, we investigated interactions between proximity, direction (approach vs. retreat), and speed during a dynamic threat-of-shock paradigm. As a measure of threat-evoked physiological arousal, skin conductance responses were recorded during fMRI scanning. Some brain regions tracked individual threat-related factors, and others were also sensitive to combinations of these variables. In particular, signals in the anterior insula tracked the interaction between proximity and direction where approach versus retreat responses were stronger when threat was closer compared with farther. A parallel proximity-by-direction interaction was also observed in physiological skin conductance responses. In the right amygdala, we observed a proximity by direction interaction, but intriguingly in the opposite direction as the anterior insula; retreat versus approach responses were stronger when threat was closer compared with farther. In the right bed nucleus of the stria terminalis, we observed an effect of threat proximity, whereas in the right periaqueductal gray/midbrain we observed an effect of threat direction and a proximity by direction by speed interaction (the latter was detected in exploratory analyses but not in a voxelwise fashion). Together, our study refines our understanding of the brain mechanisms involved during aversive anticipation in the human brain. Importantly, it emphasizes that threat processing should be understood in a manner that is both context-sensitive and dynamic.

Macrophage Recycling of Red Blood Cells and Iron Following Transfusion

Red blood cells (RBCs) destined for transfusion can be refrigerator stored for up to 42 days prior to transfusion. During this ex vivo storage period, RBCs are progressively damaged in a process that is termed the RBC storage lesion. The end result of this storage lesion is that with longer durations of storage prior to transfusion, an increased number of storage-damaged RBCs are cleared from the circulation via extravascular hemolysis. Studies in mice, dogs, and humans suggest that transfusion of older, stored RBCs, but not fresh RBCs, produce acute elevations in circulating markers of extravascular hemolysis (e.g., indirect bilirubin and transferrin saturation). Furthermore, hepcidin is induced in humans approximately 4-6 hours after transfusion and likely results in the return to baseline serum iron levels by 20-hours following transfusion. This is associated with a concomitant rise in serum ferritin levels, suggesting that hepcidin-induced degradation of ferroportin results in intracellular storage of iron within macrophages. Furthermore, the rate at which the macrophages release iron can be greater than the capacity of circulating transferrin to handle that iron, resulting in production of non-transferrin-bound iron. This pathogenic form of iron may be a cause of adverse effects in transfusion recipients. Indeed, in animal models, iron availability is associated with increased morbidity and mortality from infectious challenges with certain bacteria. A human study comparing transfusion of a single unit of RBCs following 1, 2, 3, 4, 5, or 6 weeks of storage suggests that the RBC storage lesion does not develop linearly and is only capable of inducing non-transferrin-bound iron production in healthy adult recipients following 5-6 weeks of storage. However, this relationship may differ in critically-ill populations. Furthermore, there is significant variability among blood donors in their RBC quality following storage. There are likely to be both genetic and environmental causes to this variability. Some of these causes may be donor glucose-6-phosphate dehydrogenase status, iron status, and/or dietary lipid content. In addition, in animal models, the clearance of a bolus of storage-damaged RBCs is associated with a pro-inflammatory cytokine response. Although this has yet to be demonstrated in studies using healthy adult human volunteers, studies in pediatric patient populations suggest that RBC transfusions may be associated with a pro-inflammatory response in this vulnerable population. The precise mechanism of inflammation following transfusion, and its clinical implications, has yet to be resolved. Finally, recent randomized clinical trials suggest that there are no clinical outcome differences when patients are randomized to receiving transfusions of fresh RBCs as opposed to the standard of care. Perhaps the RBC storage lesion has no clinical consequences to the transfusion recipient. However, these studies were not powered nor designed to test the effects of transfusion of RBCs during the final week of storage, in which studies suggest the effects would be most striking. Furthermore, many of the recipients in these studies already suffered from baseline hemolysis (e.g., from cardiac pump induced hemolysis, malaria, and sickle cell disease). It is unlikely that the added hemolysis from a transfusion of older RBCs would have a clinical impact given the level of baseline hemolysis from other causes observed in these patient populations. Thus, whether there are pathophysiologic consequences to the clearance of storage-damaged RBCs is still debatable. DisclosuresNo relevant conflicts of interest to declare.

The diversity of the proline-rich domain of pneumococcal surface protein A (PspA): Potential relevance to a broad-spectrum vaccine

Pneumococcal surface protein A (PspA) is a surface exposed, highly immunogenic protein of Streptococcus pneumoniae. Its N-terminal α-helical domain (αHD) elicits protective antibody in humans and animals that can protect mice from fatal infections with pneumococci and can be detected in vitro with opsonophagocytosis assays. The proline-rich domain (PRD) in the center of the PspA sequence can also elicit protection. This study revealed that although the sequence of PRD was diverse, PRD from different pneumococcal isolates contained many shared elements. The inferred amino acid sequences of 123 such PRDs, which were analyzed by assembly and alignment-free (AAF) approaches, formed three PRD groups. Of these sequences, 45 were classified as Group 1, 19 were classified as Group 2, and 59 were classified as Group 3. All Group 3 sequences contained a highly conserved 22-amino acid non-proline block (NPB). A significant polymorphism was observed, however, at a single amino acid position within NPB. Each of the three PRD groups had characteristic patterns of short amino acid repeats, with most of the repeats being found in more than one PRD group. One of these repeats, PKPEQP as well as the NPB were previously shown to elicit protective antibodies in mice. In this study, we found that sera from 12 healthy human adult volunteers contained antibodies to all three PRD groups. This suggested that a PspA-containing vaccine containing carefully selected PRDs and αHDs could redundantly cover the known diversity of PspA. Such an approach might reduce the chances of PspA variants escaping a PspA vaccine’s immunity.

Gastric cooling and menthol cause an increase in cardiac parasympathetic efferent activity in healthy adult human volunteers

What is the central question of this study? How do gastric stretch and gastric cooling stimuli affect cardiac autonomic control? What is the main finding and its importance? Gastric stretch causes an increase in cardiac sympathetic activity. Stretch combined with cold stimulation result in an elimination of the sympathetic response to stretch and an increase in cardiac parasympathetic activity, in turn resulting in a reduction in heart rate. Gastric cold stimulation causes a shift in sympathovagal balance towards parasympathetic dominance. The cold-induced bradycardia has the potential to decrease cardiac workload, which might be significant in individuals with cardiovascular pathologies. Gastric distension increases blood pressure and heart rate in young, healthy humans, but little is known about the effect of gastric stretch combined with cooling. We used a randomized crossover study to assess the cardiovascular responses to drinking 300ml of ispaghula husk solution at either 6 or 37°C in nine healthy humans (age 24.08±9.36years) to establish the effect of gastric stretch with and without cooling. The effect of consuming peppermint oil capsules to activate cold thermoreceptors was also investigated. The ECG, respiratory movements and continuous blood pressure were recorded during a 5min baseline period, followed by a 115min post-drink period, during which 5min epochs of data were recorded. Cardiac autonomic activity was assessed using time and frequency domain analyses of respiratory sinus arrhythmia to quantify parasympathetic autonomic activity, and corrected QT (QTc) interval analysis to quantify sympathetic autonomic activity. Gastric stretch only caused a significant reduction in QTc interval lasting up to 15min, with a concomitant but non-significant increase in heart rate, indicating an increased sympathetic cardiac tone. The additional effect of gastric cold stimulation was significantly to reduce heart rate for up to 15min, elevate indicators of cardiac parasympathetic tone and eliminate the reduction in QTc interval seen with gastric stretch only. Stimulation of gastric cold thermoreceptors with menthol also caused a significant reduction in heart rate and concomitant increase in the root mean square of successive differences. These findings indicate that gastric cold stimulation causes a shift in the sympathovagal balance of cardiac control towards a more parasympathetic dominant pattern.

Evaluation of end-tidal carbon dioxide gradient as a predictor of volume responsiveness in spontaneously breathing healthy adults

BackgroundMethods to guide fluid therapy in spontaneously breathing patients are scarce. No studies have reported the accuracy of end-tidal CO2 (ET-CO2) to predict volume responsiveness in these patients. We sought to evaluate the ET-CO2 gradient (ΔET-CO2) after a passive leg rise (PLR) maneuver to predict volume responsiveness in spontaneously breathing healthy adults.MethodsWe conducted a prospective study in healthy adult human volunteers. A PLR maneuver was performed and cardiac output (CO) was measured by transthoracic echocardiography. ET-CO2 was measured with non-invasive capnographs. Volume responsiveness was defined as an increase in cardiac output (CO) > 12% at 90 s after PLR.ResultsOf the 50 volunteers, 32% were classified as volume responders. In this group, the left ventricle outflow tract velocity time integral (VTILVOT) increased from 17.9 ± 3.0 to 20.4 ± 3.4 (p = 0.0004), CO increased from 4.4 ± 1.5 to 5.5 ± 1.6 (p = 0.0), and ET-CO2 rose from 32 ± 4.84 to 33 ± 5.07 (p = 0.135). Within the entire population, PLR-induced percentage ∆CO was not correlated with percentage ∆ET-CO2 (R2 = 0.13; p = 0.36). The area under the receiver operating curve for the ability of ET-CO2 to discriminate responders from non-responders was of 0.67 ± 0.09 (95% CI 0.498–0.853). A ΔET-CO2 ≥ 2 mmHg had a sensitivity of 50%, specificity of 97.06%, positive likelihood ratio of 17.00, negative likelihood ratio of 0.51, positive predictive value of 88.9%, and negative predictive value of 80.5% for the prediction of fluid responsiveness.ConclusionsΔET-CO2 after a PLR has limited utility to discriminate responders from non-responders among healthy spontaneously breathing adults.

Safety, Tolerability and Bioequivalence Assessment of a Dispersible Tablet Formulation of Pyrazinamide

Pyrazinamide is a widely used drug for the treatment of tuberculosis. A 150 mg Dispersible Tablet (DT) formulation of the drug was prepared by Micro Labs, a generic drug company. The formulation was developed for easy administration into paediatric patients. It was tested for bioequivalence in healthy adult human volunteers in fasting condition. A total of 36 subjects were enrolled in the study and all the 36 subjects completed both the two periods of the study without showing major adverse events. The pharmacokinetic parameters determined for bioequivalence were Cmax and AUC0-t. The two parameters were within the limit of bioequivalence criteria. The 150 mg DT formulation developed by Micro Labs was found to be bioequivalent to 500 mg immediate release tablet formulation of Riemser Pharma. Both the formulations were safe and well tolerated among the tested human subjects. No major adverse events were observed.

Safety and immunogenicity of a mutagenized, live attenuated Rift Valley fever vaccine, MP-12, in a Phase 1 dose escalation and route comparison study in humans

Rift Valley fever (RVF) poses a risk as a potential agent in bioterrorism or agroterrorism. A live attenuated RVF vaccine (RVF MP-12) has been shown to be safe and protective in animals and showed promise in two initial clinical trials. In the present study, healthy adult human volunteers (N=56) received a single injection of (a) RVF MP-12, administered subcutaneously (SQ) at a concentration of 104.7 plaque-forming units (pfu) (SQ Group); (b) RVF MP-12, administered intramuscularly (IM) at 103.4pfu (IM Group 1); (c) RVF MP-12, administered IM at 104.4pfu (IM Group 2); or (d) saline (Placebo Group). The vaccine was well tolerated by volunteers in all dose and route groups. Infrequent and minor adverse events were seen among recipients of both placebo and RVF MP-12. One subject had viremia detectable by direct plaque assay, and six subjects from IM Group 2 had transient low-titer viremia detectable only by nucleic acid amplification. Of the 43 vaccine recipients, 40 (93%) achieved neutralizing antibodies (measured as an 80% plaque reduction neutralization titer [PRNT80]) as well as RVF-specific IgM and IgG. The highest peak geometric mean PRNT80 titers were observed in IM Group 2. Of 34 RVF MP-12 recipients available for testing 1 year following inoculation, 28 (82%) remained seropositive (PRNT80≥1:20); this included 20 of 23 vaccinees (87%) from IM Group 2. The live attenuated RVF MP-12 vaccine was safe and immunogenic at the doses and routes studied. Given the need for an effective vaccine against RVF virus, further evaluation in humans is warranted.

Pharmacokinetic scaling of epirubicin using allometric and species-invariant time methods

The pharmacokinetics of epirubicin, an anthracycline, were investigated after intravenous bolus administration (5 mg/kg) in mice, rats, rabbits and dogs. Based on animal data, we predicted the following human pharmacokinetic parameters using allometric scaling: 120 and 35.2 L/h for total body clearance (CLt) using simple and maximum life-span potential (MLP)-corrected allometry, respectively; 702 L for steady-state volume of distribution (Vdss). The scaled Vdss value was twofold lower than the corresponding values in humans. However, the scaled CLt values were consistent with those clinically observed in humans (35.6–133.4 L/h). We also predicted human parameters using species-invariant time transformations (equivalent time, kallynochrons, apolysichrons and dienetichrons). The mean Vdss (854 L) obtained using kallynochrons and that derived from simple allometry were comparable. The lowest CLt (121 L/h) derived using kallynochrons was comparable to that obtained using simple allometry. The results of this study also indicated that the predicted human CLt generated using MLP-corrected allometry can be used for the selection of a safe dose for studies in healthy adult human volunteers. These results suggest that such approaches may be useful in designing pharmacokinetic studies for novel anthracyclines.

Red blood cell transfusion-induced inflammation: myth or reality.

Red blood cells (RBCs) destined for transfusion can be refrigerator stored for up to 42 days prior to transfusion. Our studies in mice and dogs suggest that transfusion of older, stored RBCs, but not fresh RBCs, produce acute elevations in circulating pro-inflammatory cytokine levels. Although our study in healthy adult human volunteers failed to demonstrate a pro-inflammatory cytokine response following transfusion of RBCs stored for 40-42 days, a recent study in preterm infants suggests that RBC transfusions are associated with a pro-inflammatory response. Thus, whether RBC transfusions, particularly of older, stored RBCs, result in a pro-inflammatory cytokine response, is still an unresolved issue. Animal studies suggest this to be true, yet human studies have yet to demonstrate definitively that such an inflammatory response occurs. Potential explanations for this include differences between human and animal biology, the dose of RBCs transfused, and baseline differences in levels of inflammation. This review will summarize the currently available evidence and approaches to resolving whether transfusions of older, stored RBCs are associated with inflammation in recipients.

Enantioselective Pharmacokinetics of Primaquine in Healthy Human Volunteers

Primaquine (PQ), a racemic drug, is the only treatment available for radical cure of relapsing Plasmodium vivax malaria and blocking transmission of P. falciparum malaria. Recent studies have shown differential pharmacologic and toxicologic profiles of individual PQ enantiomers in rodent, dog, and primate animal models. This study was conducted in six healthy adult human volunteers to determine the plasma pharmacokinetic profile of enantiomers of PQ and carboxyprimaquine (cPQ), the major plasma metabolite. The individuals were orally administered PQ diphosphate, equivalent to 45-mg base, 30 minutes after a normal breakfast. Blood samples were collected at different time intervals, and plasma samples were analyzed for enantiomers of PQ and cPQ. Plasma PQ concentrations were low and variable for both parent enantiomers and peaked around 2-4 hours. Peak (-)-(R)-PQ concentrations ranged from 121 ng/ml to 221 ng/ml, and peak (+)-(S)-PQ concentrations ranged from 168 ng/ml to 299 ng/ml. The cPQ concentrations were much higher and were surprisingly consistent from subject to subject. Essentially all the cPQ detected in plasma was (-)-cPQ. The peak concentrations of (-)-cPQ were observed at 8 hours (range: 1104-1756 ng/ml); however, very high concentrations were sustained through 24 hours. (+)-cPQ was two orders of magnitude lower than (-)-cPQ, and in a few subjects it was detected but only under the limit of quantification. In vitro studies with primary human hepatocytes also suggested more rapid metabolism of (-)-PQ compared with (+)-PQ. The results suggest more rapid metabolism of (-)-PQ to (-) cPQ compared with (+)-PQ. Alternatively, (+)-PQ or (+)-cPQ could be rapidly converted to another metabolite(s) or distributed to tissues. This is the first clinical report on enantioselective pharmacokinetic profiles of PQ and cPQ and supports further clinical evaluation of individual PQ enantiomers.

Self-navigated isotropic three-dimensional cardiac T2 mapping.

To implement and characterize an isotropic three-dimensional cardiac T2 mapping technique. A self-navigated three-dimensional radial segmented balanced steady-state free precession pulse sequence with an isotropic 1.7-mm spatial resolution was implemented at 3T with a variable T2 preparation module. Bloch equation and Monte Carlo simulations were performed to determine the influence of the heart rate, B1 inhomogeneity and noise on the T2 fitting accuracy. In a phantom study, the accuracy of the pulse sequence was studied through comparison with a gold-standard spin-echo T2 mapping method. The robustness and homogeneity of the technique were ascertained in a study of 10 healthy adult human volunteers, while first results obtained in patients are reported. The numerical simulations demonstrated that the heart rate and B1 inhomogeneity cause only minor deviations in the T2 fitting, whereas the phantom study showed good agreement of the technique with the gold standard. The volunteer study demonstrated an average myocardial T2 of 40.5 ± 3.3 ms and a <15% T2 gradient in the base-apex and anterior-inferior direction. In three patients, elevated T2 values were measured in regions with expected edema. This respiratory self-navigated isotropic three-dimensional technique allows for accurate and robust in vitro and in vivo T2 quantification.

Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects.

Ozone (O3) is a criteria air pollutant that is associated with numerous adverse health effects, including altered respiratory immune responses. Despite its deleterious health effects, possible epigenetic mechanisms underlying O3-induced health effects remain understudied. MicroRNAs (miRNAs) are epigenetic regulators of genomic response to environmental insults and unstudied in relationship to O3 inhalation exposure. Our objective was to test whether O3 inhalation exposure significantly alters miRNA expression profiles within the human bronchial airways. Twenty healthy adult human volunteers were exposed to 0.4 ppm O3 for 2 h. Induced sputum samples were collected from each subject 48 h preexposure and 6 h postexposure for evaluation of miRNA expression and markers of inflammation in the airways. Genomewide miRNA expression profiles were evaluated by microarray analysis, and in silico predicted mRNA targets of the O3-responsive miRNAs were identified and validated against previously measured O3-induced changes in mRNA targets. Biological network analysis was performed on the O3-associated miRNAs and mRNA targets to reveal potential associated response signaling and functional enrichment. Expression analysis of the sputum samples revealed that O3 exposure significantly increased the expression levels of 10 miRNAs, namely miR-132, miR-143, miR-145, miR-199a*, miR-199b-5p, miR-222, miR-223, miR-25, miR-424, and miR-582-5p. The miRNAs and their predicted targets were associated with a diverse range of biological functions and disease signatures, noted among them inflammation and immune-related disease. The present study shows that O3 inhalation exposure disrupts select miRNA expression profiles that are associated with inflammatory and immune response signaling. These findings provide novel insight into epigenetic regulation of responses to O3 exposure.

Application of Microarray and Functional-Based Screening Methods for the Detection of Antimicrobial Resistance Genes in the Microbiomes of Healthy Humans

The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), bla TEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.

Infusion of Pharmaceutical-Grade Natural Human C-Reactive Protein Is Not Proinflammatory in Healthy Adult Human Volunteers

Baseline circulating concentrations of C-reactive protein (CRP) are significantly associated with cardiovascular disease risk in general populations. This modest association has been inappropriately conflated with causality, and it has been claimed that CRP is proatherogenic. Most of the known causative factors for atherosclerosis stimulate increased CRP production, but comprehensive genetic epidemiology studies provide no support for a pathogenic role of CRP. The reported proinflammatory effects of human CRP preparations on healthy cells in vitro and in healthy animals in vivo have all been produced by poorly characterized CRP preparations, demonstrably caused by impurities, or elicited by CRP made in recombinant Escherichia coli not by humans. None of the in vitro or animal findings have been reproduced with pure natural human CRP. Nevertheless, the strong proinflammatory effects of infusing recombinant bacterial CRP into humans have still been inappropriately ascribed to CRP. To investigate the effects of infusion into healthy adult human volunteers of pure natural human CRP. Comprehensively characterized, pharmaceutical-grade, endotoxin-free, purified CRP, prepared to GMP standard from pooled normal human donor plasma was infused as an intravenous bolus in 7 healthy adult human volunteers at ≤2 mg/kg to provide circulating CRP concentrations ≤44 mg/L. No recipient showed any significant clinical, hematologic, coagulation, or biochemical changes, or any increase in proinflammatory cytokines or acute phase proteins. The human CRP molecule itself is not proinflammatory in healthy human adults.

Prograf Five Milligrams Versus Tacrolimus Medis in Healthy Volunteers: A Bioequivalence Study

For FDA approval, bioequivalence of a generic version of Tacrolimus must be demonstrated in a randomized, two-treatments, two-periods, two-sequences, single-dose crossover study in healthy adult volunteers. Currently there are at least 3 differents generic equivalent for Tacrolimus, that are approved by the EMA and the FDA, with a USA market share of nearly 50%. However, the market share of generic immunosuppressive drugs in the Middle East region is still very low due to the reluctance of the physician to accept Tacrolimus generics, considered to be a narrow therapeutic window drug, that are approved using the standard bioequivalence criteria of 80% to 125%. Herein we present a bioequivalence study of a new Tacrolimus generic, Tacrolimus Medis 5 mg developed by Medis Tunisia batch number 12G3003 compared with Prograf® 5 mg batch number 7202 manufactured by Astellas Toyama Co., Ltd. Japan and HIKMA Pharmaceuticals, Amman-Jordan in healthy adult volunteers using the 90%–111% criteria recommended for drugs with narrow therapeutic window. The study was, balanced, randomized, two-treatments, two-periods, two-sequences, single dose, crossover, comparative oral bioavailability study in healthy adult human volunteers. The study was carried out in accordance with the Basic Principles defined in the U.S. 21 CFR Part 312.20, the principles enunciated in the Declaration of Helsinki (World Medical Association Declaration of Helsinki). Thirty six non-smoking healthy, as determined by medical history, volunteers, 18 years and older, were included. Following randomization using a computer software (pharma solution) the volunteers were given a single oral dose of 5 milligrams following a 12 hour fast with a wash out period of 7 days. Pharmacokinetics profile with blood levels at: 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, and 24 hours were performed following each dose. Tacrolimus plasma level was determined using an HPLC validated method (Transmedical For Life S.A.R.L. Beirut Lebanon), for accuracy, suitability, reproducibility, precision , long-term stability and robustness. Physical examinations, hematology, urine analysis and serum chemistry tests were performed at screening and before dosing in each period and at end of the study. Volunteers were monitored for safety and adverse events throughout the study. Both products were bioequivalent at the entire pharmacokinetic parameters tested. The LSM were 95.31%–101.21% for AUC, 94.65%–101.11% for AUC0-inf, 97.15%–100.02% for Cmax and 91.54%–103.75% for Half-life. Respectively all of which are within the EU and FDA approval limits (90–111%) indicating that the 2 products are equivalent and switchable.