Characterization and first-in-human clinical dose-escalation safety evaluation of a next-gen human freeze-dried plasma.

BACKGROUND: Early plasma transfusion is life-saving for bleeding trauma patients. Freeze-dried plasma (FDP) provides unique formulation advantages for infusion in the prehospital setting. We describe characterization and clinical safety data of the first, next-generation FDP stored in plastic bags with rapid reconstitution. STUDY DESIGN AND METHODS: Coagulation and chemistry parameters on 155 pairs of fresh frozen plasma (FFP) and their derivative FDP units were compared. Next, a first-in-human, dose-escalation safety evaluation of FDP, involving 24 healthy volunteers who donated either whole blood or apheresis plasma to create autologous FDP, was performed in three dose cohorts (270, 540, and 810 ml) and adverse events (AEs) were monitored. Cohort 3 was randomized, double-blind with a cross-over arm that compared FDP versus FFP using descriptive analysis for AEs, coagulation, hematology, and chemistry parameters. RESULTS: FDP coagulation factors, clotting times, and product quality (pH, total protein, and osmolality) post-lyophilization were preserved. FDP infusions, of up to 810 ml per subject, were found to be safe and with no serious AEs (SAEs) related to FDP. The average time to reconstitute FDP was 67 s (range: 43-106). No differences in coagulation parameters or thrombin activation were detected in subjects infused with 810 ml of FDP compared with FFP. CONCLUSION: This first next-generation FDP product preserves the potency and safety of FFP in a novel rugged, compressible, plastic container, for rapid transfusion, allowing rapid access to plasma in resuscitation protocols for therapy in acute traumatic hemorrhage.

An Analysis of Ketamine Doses Administrated to Nonintubated Casualties Prehospital.

INTRODUCTION: Previous studies demonstrate that a significant proportion of casualties do not receive pain medication prehospital after traumatic injuries. To address possible reasons, the U.S. Military has sought to develop novel delivery methods to aid in administration of pain medications prehospital. We sought to describe the dose and route of ketamine administered prehospital to help inform materiel solutions. MATERIALS AND METHODS: This is a secondary analysis of a previously described dataset focused on prehospital data within the Department of Defense Trauma Registry from 2007 to 2020. We isolated encounters in which ketamine was administered along with the amount dosed and the route of administration in nonintubated patients. RESULTS: Within our dataset, 862 casualties met inclusion for this analysis. The median age was 28 and nearly all (98%) were male. Most were battle injuries (88%) caused by explosives (54%). The median injury severity score was 10 with the extremities accounting to the most frequent seriously injured body region (38%). The mean dose via intravenous route was 50.4 mg (n = 743, 95% CI 46.5-54.3), intramuscular was 66.7 mg (n = 234, 95% CI 60.3-73.1), intranasal was 56.5 mg (n = 10, 39.1-73.8), and intraosseous was 83.3 mg (n = 34, 66.3-100.4). Most had a medic or CLS in their chain of care (87%) with air evacuation as the primary mechanism of evacuation (86%). CONCLUSIONS: The average doses administered were generally larger than the doses recommended by Tactical Combat Casualty Care guidelines. Currently, guidelines may underdose analgesia. Our data will help inform materiel solutions based on end-user requirements.

Micro-RNA 133a-3p induces repolarization abnormalities in atrial myocardium and modulates ventricular electrophysiology affecting ICa,L and Ito currents.

Mir-133a-3p is the most abundant myocardial microRNA. The impact of mir-133a-3p on cardiac electrophysiology is poorly explored. In this study, we investigated the effects of mir-133a-3p on the main ionic currents critical for action potential (AP) generation and electrical activity of the heart. We used conventional ECG, sharp microelectrodes and patch-clamp to clarify a role of mir-133a-3p in normal cardiac electrophysiology in rats after in vivo and in vitro transfection. Mir-133a-3p caused no changes to pacemaker APs and automaticity in the sinoatrial node. No significant changes in heart rate (HR) were observed in vivo; however, miR transfection facilitated HR increase in response to ß-adrenergic stimulation. Mir-133a-3p induced repolarization abnormalities in the atrial working myocardium and the L-type calcium current (ICa,L) was significantly increased. The main repolarization currents, including the transient outward (Ito), ultra-rapid (IK,ur), and inward rectifier (IK1) remained unaffected in atrial cardiomyocytes. Mir-133a-3p affected both ICa,L and Ito in ventricular cardiomyocytes. Systemic administration of mir-133a-3p induced QT-interval prolongation. Bioinformatic analysis revealed protein phosphatase 2 (PPP2CA/B) and Kcnd3 (encoding Kv4.3 channels generating Ito) as the main miR-133a-3p targets in the heart. No changes in mRNA expression of Cacna1c (encoding Cav1.2 channels generating ICa,L) and Kcnd3 were seen in mir-133a-3p treated rats. However, the expression of Ppp2cA, encoding PPP2CA, and Kcnip2 encoding KChIP2, a Kv4.3 regulatory protein, were significantly decreased. The accumulation of mir-133a-3p in cardiac myocytes causes chamber-specific electrophysiological changes. The suppression of PPP2CA, involved in adrenergic signal transduction, and Kchip2 may indirectly mediate mir-133a-3p-induced augmentation of ICa,L and attenuation of Ito.

Further insights into the molecular complexity of the human sinus node - The role of 'novel' transcription factors and microRNAs.

RESEARCH PURPOSE: The sinus node (SN) is the heart's primary pacemaker. Key ion channels (mainly the funny channel, HCN4) and Ca2+-handling proteins in the SN are responsible for its function. Transcription factors (TFs) regulate gene expression through inhibition or activation and microRNAs (miRs) do this through inhibition. There is high expression of macrophages and mast cells within the SN connective tissue. 'Novel'/unexplored TFs and miRs in the regulation of ion channels and immune cells in the SN are not well understood. Using RNAseq and bioinformatics, the expression profile and predicted interaction of key TFs and cell markers with key miRs in the adult human SN vs. right atrial tissue (RA) were determined. PRINCIPAL RESULTS: 68 and 60 TFs significantly more or less expressed in the SN vs. RA respectively. Among those more expressed were ISL1 and TBX3 (involved in embryonic development of the SN) and 'novel' RUNX1-2, CEBPA, GLI1-2 and SOX2. These TFs were predicted to regulate HCN4 expression in the SN. Markers for different cells: fibroblasts (COL1A1), fat (FABP4), macrophages (CSF1R and CD209), natural killer (GZMA) and mast (TPSAB1) were significantly more expressed in the SN vs. RA. Interestingly, RUNX1-3, CEBPA and GLI1 also regulate expression of these cells. MiR-486-3p inhibits HCN4 and markers involved in immune response. MAJOR CONCLUSIONS: In conclusion, RUNX1-2, CSF1R, TPSAB1, COL1A1 and HCN4 are highly expressed in the SN but not miR-486-3p. Their complex interactions can be used to treat SN dysfunction such as bradycardia. Interestingly, another research group recently reported miR-486-3p is upregulated in blood samples from severe COVID-19 patients who suffer from bradycardia.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Corneal confocal microscopy identifies small fibre damage and progression of diabetic neuropathy.

Accurately quantifying the progression of diabetic peripheral neuropathy is key to identify individuals who will progress to foot ulceration and to power clinical intervention trials. We have undertaken detailed neuropathy phenotyping to assess the longitudinal utility of different measures of neuropathy in patients with diabetes. Nineteen patients with diabetes (age 52.5 ± 14.7 years, duration of diabetes 26.0 ± 13.8 years) and 19 healthy controls underwent assessment of symptoms and signs of neuropathy, quantitative sensory testing, autonomic nerve function, neurophysiology, intra-epidermal nerve fibre density (IENFD) and corneal confocal microscopy (CCM) to quantify corneal nerve fibre density (CNFD), branch density (CNBD) and fibre length (CNFL). Mean follow-up was 6.5 years. Glycated haemoglobin (p = 0.04), low-density lipoprotein-cholesterol (LDL-C) (p = 0.0009) and urinary albumin creatinine ratio (p < 0.0001) improved. Neuropathy symptom profile (p = 0.03), neuropathy disability score (p = 0.04), vibration perception threshold (p = 0.02), cold perception threshold (p = 0.006), CNFD (p = 0.03), CNBD (p < 0.0001), CNFL (p < 0.0001), IENFD (p = 0.04), sural (p = 0.02) and peroneal motor nerve conduction velocity (p = 0.03) deteriorated significantly. Change (∆) in CNFL correlated with ∆CPT (p = 0.006) and ∆Expiration/Inspiration ratio (p = 0.002) and ∆IENFD correlated with ∆CNFD (p = 0.005), ∆CNBD (p = 0.02) and ∆CNFL (p = 0.01). This study shows worsening of diabetic neuropathy across a range of neuropathy measures, especially CCM, despite an improvement in HbA1c and LDL-C. It further supports the utility of CCM as a rapid, non-invasive surrogate measure of diabetic neuropathy.

Identification of Key Small Non-Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node.

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca2+-handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Cav)1.3, and Cav3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% (P<0.05) and HCN4 expression (P<0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Sinus node-like pacemaker mechanisms regulate ectopic pacemaker activity in the adult rat atrioventricular ring.

In adult mammalian hearts, atrioventricular rings (AVRs) surround the atrial orifices of atrioventricular valves and are hotbed of ectopic activity in patients with focal atrial tachycardia. Experimental data offering mechanistic insights into initiation and maintenance of ectopic foci is lacking. We aimed to characterise AVRs in structurally normal rat hearts, identify arrhythmia predisposition and investigate mechanisms underlying arrhythmogenicity. Extracellular potential mapping and intracellular action potential recording techniques were used for electrophysiology, qPCR for gene and, Western blot and immunohistochemistry for protein expression. Conditions favouring ectopic foci were assessed by simulations. In right atrial preparations, sinus node (SN) was dominant and AVRs displayed 1:1 impulse conduction. Detaching SN unmasked ectopic pacemaking in AVRs and pacemaker action potentials were SN-like. Blocking pacemaker current If, and disrupting intracellular Ca2+ release, prolonged spontaneous cycle length in AVRs, indicating a role for SN-like pacemaker mechanisms. AVRs labelled positive for HCN4, and SERCA2a was comparable to SN. Pacemaking was potentiated by isoproterenol and abolished with carbachol and AVRs had abundant sympathetic nerve endings. ß2-adrenergic and M2-muscarinic receptor mRNA and ß2-receptor protein were comparable to SN. In computer simulations of a sick SN, ectopic foci in AVR were unmasked, causing transient suppression of SN pacemaking.

A sexy approach to pacemaking: differences in function and molecular make up of the sinoatrial node.

BACKGROUND: Functional properties of the sinoatrial node (SAN) are known to differ between sexes. Women have higher resting and intrinsic heart rates. Sex determines the risk of developing certain arrhythmias such as sick sinus syndrome, which occur more often in women. We believe that a major contributor to these differences is in gender specific ion channel expression. METHODS: qPCR was used to compare ion channel gene expression in the SAN and right atrium (RA) between male and female rats. Histology, immunohistochemistry and signal intensity analysis were used to locate the SAN and determine abundance of ion channels. The effect of nifedipine on extracellular potential recording was used to determine differences in beating rate between sexes. RESULTS: mRNAs for Cav1.3, Kir3.1, and Nkx2-5, as well as expression of the L-Type Ca²âº channel protein, were higher in the female SAN. Females had significantly higher intrinsic heart rates and the effect of nifedipine on isolated SAN preparations was significantly greater in male SAN. Computer modelling using a SAN cell model demonstrated a higher propensity of pacemaker-related arrhythmias in females. CONCLUSION: This study has identified key differences in the expression of Cav1.3, Kir3.1 and Nkx2-5 at mRNA and/or protein levels between male and female SAN. Cav1.3 plays an important role in the pacemaker function of the SAN, therefore the higher intrinsic heart rate of the female SAN could be caused by the higher expression of Cav1.3. The differences identified in this study advance our understanding of sex differences in cardiac electrophysiology and arrhythmias.

Ca(2+)-Clock-Dependent Pacemaking in the Sinus Node Is Impaired in Mice with a Cardiac Specific Reduction in SERCA2 Abundance.

BACKGROUND: The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) pump is an important component of the Ca(2+)-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2's role in sinus node pacemaking. METHODS AND RESULTS: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34% (all P < 0.05 vs. Serca2 FF) lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs(+) caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, I f. Disabling the Ca(2+)-clock with 2 µM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations (9 ± 1% vs. 20 ± 3% in Serca2 FF; P < 0.05), suggesting a disrupted Ca(2+)-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca(2+)-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity. CONCLUSIONS: Serca2 KO mice show a disrupted Ca(2+)-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function.

US Army blood program: 2025 and beyond.

In preparing to support the Army in 2025 and beyond, the Army Blood Program remains actively engaged with the research and advanced development of blood products and medical technology to improve blood safety and efficacy in conjunction with the US Army Medical Research and Materiel Command. National and International Blood Bank authorities have noted that the US Army research and development efforts in providing new blood products and improving blood safety operate on the cutting edge of technology and are transformational for the global blood industry. Over the past 14 years, the Army has transformed how blood support is provided and improved the survival rate of casualties. Almost every product or process developed by or for the military has found an application in treating civilian patients. Conflicts have many unwanted consequences; however, in times of conflict, one positive aspect is the identification of novel solutions to improve the safety and efficacy of the blood supply.

3D anatomical reconstruction of human cardiac conduction system and simulation of bundle branch block after TAVI procedure.

The cardiac conduction system (CCS) is responsible for the initiation and propagation of action potentials through the heart ensuring efficient pumping of blood. Understanding the anatomy of the CCS and its relationship with other major cardiac components is important to help understand arrhythmias and how certain procedures may increase the incidence of arrhythmias developing. We sectioned a whole human heart and performed Masson's trichrome histology in order to identify the components of the CCS. The histology images were used to construct a 3D anatomical model. We have shown that it is possible to create a 3D anatomical model of the human heart incorporating the CCS based on histological images, and that this model can be used to perform computer simulations of cardiac excitation. From the reconstruction we have been able to show the relative positions of the CCS components to each other. We have also shown how the close proximity of the CCS to the aortic valve can explain some of the conduction complications, such as left bundle branch block, that arise after aortic valve replacement procedures.

Congestive Heart Failure Leads to Prolongation of the PR Interval and Atrioventricular Junction Enlargement and Ion Channel Remodelling in the Rabbit.

Heart failure is a major killer worldwide. Atrioventricular conduction block is common in heart failure; it is associated with worse outcomes and can lead to syncope and bradycardic death. We examine the effect of heart failure on anatomical and ion channel remodelling in the rabbit atrioventricular junction (AVJ). Heart failure was induced in New Zealand rabbits by disruption of the aortic valve and banding of the abdominal aorta resulting in volume and pressure overload. Laser micro-dissection and real-time polymerase chain reaction (RT-PCR) were employed to investigate the effects of heart failure on ion channel remodelling in four regions of the rabbit AVJ and in septal tissues. Investigation of the AVJ anatomy was performed using micro-computed tomography (micro-CT). Heart failure animals developed first degree heart block. Heart failure caused ventricular myocardial volume increase with a 35% elongation of the AVJ. There was downregulation of HCN1 and Cx43 mRNA transcripts across all regions and downregulation of Cav1.3 in the transitional tissue. Cx40 mRNA was significantly downregulated in the atrial septum and AVJ tissues but not in the ventricular septum. mRNA abundance for ANP, CLCN2 and Navß1 was increased with heart failure; Nav1.1 was increased in the inferior nodal extension/compact node area. Heart failure in the rabbit leads to prolongation of the PR interval and this is accompanied by downregulation of HCN1, Cav1.3, Cx40 and Cx43 mRNAs and anatomical enlargement of the entire heart and AVJ.

Corneal Confocal Microscopy Identifies Small-Fiber Neuropathy in Subjects With Impaired Glucose Tolerance Who Develop Type 2 Diabetes.

OBJECTIVE: Impaired glucose tolerance (IGT) through to type 2 diabetes is thought to confer a continuum of risk for neuropathy. Identification of subjects at high risk of developing type 2 diabetes and, hence, worsening neuropathy would allow identification and risk stratification for more aggressive management. RESEARCH DESIGN AND METHODS: Thirty subjects with IGT and 17 age-matched control subjects underwent an oral glucose tolerance test, assessment of neuropathic symptoms and deficits, quantitative sensory testing, neurophysiology, skin biopsy, and corneal confocal microscopy (CCM) to quantify corneal nerve fiber density (CNFD), branch density (CNBD), and fiber length (CNFL) at baseline and annually for 3 years. RESULTS: Ten subjects who developed type 2 diabetes had a significantly lower CNFD (P = 0.003), CNBD (P = 0.04), and CNFL (P = 0.04) compared with control subjects at baseline and a further reduction in CNFL (P = 0.006), intraepidermal nerve fiber density (IENFD) (P = 0.02), and mean dendritic length (MDL) (P = 0.02) over 3 years. Fifteen subjects who remained IGT and 5 subjects who returned to normal glucose tolerance had no significant baseline abnormality on CCM or IENFD but had a lower MDL (P < 0.0001) compared with control subjects. The IGT subjects showed a significant decrease in IENFD (P = 0.02) but no change in MDL or CCM over 3 years. Those who returned to NGT showed an increase in CNFD (P = 0.05), CNBD (P = 0.04), and CNFL (P = 0.05), but a decrease in IENFD (P = 0.02), over 3 years. CONCLUSIONS: CCM and skin biopsy detect a small-fiber neuropathy in subjects with IGT who develop type 2 diabetes and also show a dynamic worsening or improvement in corneal and intraepidermal nerve morphology in relation to change in glucose tolerance status.

Functional, anatomical, and molecular investigation of the cardiac conduction system and arrhythmogenic atrioventricular ring tissue in the rat heart.

BACKGROUND: The cardiac conduction system consists of the sinus node, nodal extensions, atrioventricular (AV) node, penetrating bundle, bundle branches, and Purkinje fibers. Node-like AV ring tissue also exists at the AV junctions, and the right and left rings unite at the retroaortic node. The study aims were to (1) construct a 3-dimensional anatomical model of the AV rings and retroaortic node, (2) map electrical activation in the right ring and study its action potential characteristics, and (3) examine gene expression in the right ring and retroaortic node. METHODS AND RESULTS: Three-dimensional reconstruction (based on magnetic resonance imaging, histology, and immunohistochemistry) showed the extent and organization of the specialized tissues (eg, how the AV rings form the right and left nodal extensions into the AV node). Multiextracellular electrode array and microelectrode mapping of isolated right ring preparations revealed robust spontaneous activity with characteristic diastolic depolarization. Using laser microdissection gene expression measured at the mRNA level (using quantitative PCR) and protein level (using immunohistochemistry and Western blotting) showed that the right ring and retroaortic node, like the sinus node and AV node but, unlike ventricular muscle, had statistically significant higher expression of key transcription factors (including Tbx3, Msx2, and Id2) and ion channels (including HCN4, Cav3.1, Cav3.2, Kv1.5, SK1, Kir3.1, and Kir3.4) and lower expression of other key ion channels (Nav1.5 and Kir2.1). CONCLUSIONS: The AV rings and retroaortic node possess gene expression profiles similar to that of the AV node. Ion channel expression and electrophysiological recordings show the AV rings could act as ectopic pacemakers and a source of atrial tachycardia.

Human safety and pharmacokinetic study of intramuscular midazolam administered by autoinjector.

Midazolam in an autoinjector was evaluated in an open-label dose escalation study involving 39 healthy participants. Safety and pharmacokinetic parameters were determined for doses ranging from 5 to 30 mg. No serious adverse events were noted during the study. Two participants (30 mg) experienced changes in their electrocardiogram (trigeminy and prolongation of QRS complex) that met the criteria for dose-limiting adverse events. No significant respiratory depression was noted during the study. The midazolam doses studied exhibited a median t(max) of 0.5 hours with a geometric mean terminal elimination half-life value of 4.1 hours (range, 2.9-4.5 hours). The extent of systemic exposure, assessed by area under the curve (AUC) and maximum concentration (C(max)), tended to increase proportionally with increasing doses from 5 to 30 mg; however, for the male 30-mg group, there was evidence of a larger than proportional increase in AUC.

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.