Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

Background:

Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters.

Methods:

We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

Results:

TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC₅₀ of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo.Accounting for species differences in protein binding, the projected human NET and SERT plasma EC₅₀ values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [¹¹C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [¹¹C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC₅₀ for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL.

Conclusions:

These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.     

Early time course of neointima formation and vascular remodelling following percutaneous coronary intervention and vascular brachytherapy of in-stent restenotic lesions as assessed by intravascular ultrasound analysis

Summary In-stent restenosis (ISR) represents the major limitation of stent implantation. Treatment, although of relative technical ease, is unsatisfactory due to a high incidence of recurrent restenosis. Vascular brachytherapy (VBT) has emerged as a powerful adjunct therapeutic modality to treat ISR. Inhibition of neointima formation has been regarded as the relevant mechanism of action. Yet, positive remodelling has been suspected as another contributing factor. Since only very few precise analyses of the extent, distribution and time course of the respective mechanims exist, the goal of the present study was to describe the changes of the vessel geometry at the target lesion and at the reference site following angioplasty and VBT of ISR in 42 patients by means of quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS) before and after the index procedure and at the 3 and 6 month follow-up.By QCA the acute lumen gain measured 2.2±0.8 mm, the late lumen loss at 3 months was 0.1±0.5 mm and at 6 months 0.4±0.7 mm. By IVUS luminal cross-sectional area increased from 1.5±1.2 mm² to 7.9±1.9 mm² (p<0.001). The intima hyperplasia cross-sectional area at 3 months was only 0.2±1.0 mm² (p=0.191), but increased to 0.7±0.6 mm² (p<0.001) at 6 months resulting in a lumen cross-sectional area of 7.1±1.7 mm². Stent dimensions did not show any significant changes over time. The external elastic membrane cross-sectional area at 3 months increased by 1.3±1.9 mm² (p<0.001), and showed a further increase by 0.7±2.9 mm² at 6 months. Positive remodelling could be demonstrated also at the reference segment.In conclusion the absolute amount of intima hyperplasia during a 6-month follow-up period after VBT of ISR is low and most pronounced between the third and sixth month. Besides this, predominantly within the first 3 months of follow-up, significant positive remodelling could be demonstrated at the target lesion and at the reference site. Both observed effects may contribute to the preservation of the vessel lumen.Diese Publikation enthält die Ergebnisse der Promotionsarbeit von Frau Andrea Zimmermann     

Über den Glykogengehalt in Mundhöhlencarcinomen

Zusammenfassung Im Rahmen einer größeren Untersuchung über die Möglichkeiten einer Früherkennung bösartiger Tumoren in der Mundhöhle werden 22 verschiedene Mundhöhlencarcinome auf ihren Gehalt an Glykogen untersucht. Dabei ergibt sich gegenüber den Carcinomen der Portio uteri einrelativ hoher Prozentsatz glykogenhaltiger Carcinome. Vergleichsweise werden daneben die Glykogenverhältnisse der normalen menschlichen Mundschleimhaut an 432 Mundschleimhautexcisionen lebender Patienten histochemisch untersucht und ein unterschiedlicher Glykogengehalt in den einzelnen Mundhöhlenregionen aufgedeckt. Innerhallb der Epithelschichten bleibt das Stratum germinativum stets glykogenfrei. Vergleichende Untersuchungen an der glykogenfreien Mundschleimhaut von Ratten und Kaninchen konnten nachweisen, daß Teilungszellen zur Glykogensynthese nicht in der Lage sind. Zur Klärung der Frage, ob der Glykogengehalt innerhalb der Mundschleimhaut dem Insulin-Adrenalin-Regulator unterliegt, wurden darüber hinaus die Mundschleimhautverhältnisse mittels der Schillerschen Jodlösung an 1661 Diabetikern geprüft und die Mundschleimhautexcisionen von 32 Diabetikern histochemisch ausgewertet und zum jeweiligen Blutzuckerspiegel in Relation gesetzt. Die Ergebnisse sprechen dafür, daß der Glykogengehalt der Mundschleimhaut nicht vom Insulinspiegel abhängt. Da sich für Phosphorylierungsvorgänge Anhaltspunkte nicht ergaben, wird das Glykogen der Mundschleimhaut und der Mundhöhlencarcinome als Stapelungsglykogen angesprochen.     

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics,Content and Time Course of SARS-CoV-2 Neutralizing Antibodies

BackgroundConvalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma.MethodsPatients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed.ResultsA series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from <1:20 to >1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels.ConclusionWe demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.     

Comparison of acute and long-term results and underlying mechanisms from sirolimus-eluting stent implantation for the treatment of in-stent restenosis and recurrent in-stent restenosis in patients in whom intracoronary radiation failed as assessed by intravascular ultrasound

In-stent restenosis (ISR), especially after vascular brachytherapy, is a therapeutic challenge. Sirolimus-eluting stent implantation is a promising new option for the treatment of patients with ISR. The efficacy of sirolimus-eluting stent implantation for the treatment of patients with their first episodes of ISR and with recurrent ISR due to the failure of vascular brachytherapy was compared using intravascular ultrasound imaging.     

Partner choice and fidelity stabilize coevolution in a Cretaceous-age defensive symbiosis

Many insects rely on symbiotic microbes for survival, growth, or reproduction. Over evolutionary timescales, the association with intracellular symbionts is stabilized by partner fidelity through strictly vertical symbiont transmission, resulting in congruent host and symbiont phylogenies. However, little is known about how symbioses with extracellular symbionts, representing the majority of insect-associated microorganisms, evolve and remain stable despite opportunities for horizontal exchange and de novo acquisition of symbionts from the environment. Here we demonstrate that host control over symbiont transmission (partner choice) reinforces partner fidelity between solitary wasps and antibiotic-producing bacteria and thereby stabilizes this Cretaceous-age defensive mutualism. Phylogenetic analyses show that three genera of beewolf wasps (Philanthus, Trachypus, and Philanthinus) cultivate a distinct clade of Streptomyces bacteria for protection against pathogenic fungi. The symbionts were acquired from a soil-dwelling ancestor at least 68 million years ago, and vertical transmission via the brood cell and the cocoon surface resulted in host–symbiont codiversification. However, the external mode of transmission also provides opportunities for horizontal transfer, and beewolf species have indeed exchanged symbiont strains, possibly through predation or nest reuse. Experimental infection with nonnative bacteria reveals that—despite successful colonization of the antennal gland reservoirs—transmission to the cocoon is selectively blocked. Thus, partner choice can play an important role even in predominantly vertically transmitted symbioses by stabilizing the cooperative association over evolutionary timescales.Cooperation is ubiquitous in nature, yet it presents a conundrum to evolutionary biology because acts that are beneficial to the receiver but costly to the actor should not be favored by natural selection (1). In interspecific associations (i.e., symbioses), the two most important models to explain the maintenance of cooperation are partner fidelity and partner choice (2, 3). In partner-fidelity associations, host and symbiont interact repeatedly and reward cooperating individuals while punishing cheaters, thereby reinforcing mutually beneficial interactions (2, 4). In partner-choice associations, individuals may interact only once, but one member can select its partner in advance of any possible exploitation (2, 4). Partner choice appears to select for cooperative strains among environmentally acquired microbial symbionts, e.g., the bioluminescent Vibrio fischeri bacteria of squids (5), the nitrogen-fixing rhizobia of legumes (6), and mycorrhizal fungi of plants (7). By contrast, partner fidelity is generally assumed to be the major stabilizing force in the widespread and ecologically important vertically transmitted symbioses of insects (4).However, localization and transmission routes of mutualistic bacteria in insects are diverse, and the differences across symbiotic systems have important implications for the evolutionary trajectory of the associations. Symbionts with an obligate intracellular lifestyle are usually tightly integrated into the host’s metabolism (e.g., ref. 8) and development (9), and the mutual interdependence of both partners coincides with perfect vertical symbiont transmission. Over evolutionary timescales, the high degree of partner fidelity results in host–symbiont cocladogenesis, and, concordantly, phylogenies of hosts and their intracellular symbionts are often found to be congruent (10–13). Although such a pattern is also observed for some extracellular symbioses with especially tight host–symbiont integration (14, 15), the ability of many extracellularly transmitted symbionts to spend part of their life cycle outside of the host’s body is often reflected in more or less extensive horizontal transmission or de novo acquisition of symbionts from the environment (16, 17). In these cases, partner choice mechanisms are expected to ensure specificity in the establishment and maintenance of the association (18). The nature of such control mechanisms, however, remains poorly understood.Although many of the well-studied mutualistic associations in insects have a nutritional basis (19, 20), an increasing number of symbioses for the defense of the host against predators (21), parasitoids (22), or pathogens (23–25) have recently been discovered. Among defensive symbionts, Actinobacteria are particularly prevalent, probably due to their ubiquity in the soil and their ability to produce secondary metabolites with antibiotic properties (23). Antibiotic-producing actinobacterial symbionts have been discovered on the cuticle of leaf-cutting ants (26), in the fungal galleries of a bark beetle (27), and in the antennae and on cocoons of beewolf wasps (28). While in the former two cases the symbionts have been implicated in the defense of the hosts’ nutritional resources against competing fungi (26, 27), the beewolves’ bacteria protect the offspring in the cocoon against pathogenic microorganisms (28, 29).Beewolves are solitary wasps in the genera Philanthus, Trachypus, and Philanthinus (Hymenoptera, Crabronidae, Philanthini). They engage in a defensive alliance with the Actinobacterium ‘Candidatus Streptomyces philanthi’ (CaSP) (28, 30, 31), which is cultivated by female beewolves in specialized antennal gland reservoirs (32). The uniqueness and complexity of the glands suggest a long history of host adaptation towards cultivating its actinobacterial symbionts (32). From the antennae, the streptomycetes are secreted into the brood cell, taken up by the larva, and incorporated into its cocoon (33), where they provide protection against pathogenic fungi and bacteria (28) by producing at least nine different antimicrobial compounds (29). Weeks or months later, eclosing adult females acquire the bacteria from the cocoon surface (33), thus completing the vertical transmission of CaSP. However, this mode of transmission provides opportunities for the horizontal transfer of symbionts among beewolf species or the de novo uptake of bacteria from the environment. Despite these opportunities, a monophyletic clade of CaSP strains has previously been found in 31 species of beewolves, suggesting an ancient and highly coevolved relationship (30, 31, 34).Here we combine cophylogenetic analyses of beewolves and their vertically transmitted defensive symbionts with experimental manipulation of symbiont infection status and subsequent observations of transmission from female antennal gland reservoirs into the brood cell to (i) reconstruct the coevolutionary history of the symbiosis, (ii) estimate the age of the symbiosis, (iii) elucidate the ancestral lifestyle of the symbionts, and (iv) assess the importance of partner fidelity and partner choice for the long-term stability of the association.