Isostearic acid is an active component of imiquimod formulations used to induce psoriaform disease models.

Topical imiquimod based creams are indicated as immune stimulants for papillomas and various skin neoplasms. Imiquimod is considered a TLR7 ligand. These creams are also used in research to induce skin inflammation in mice as a model for psoriasis. We observed that this inflammatory response was not strictly imiquimod dependent and we set out to establish which components drive the proinflammatory effects. To this end, we examined the induction response in a BALB/cJRj mouse model, in which 50 mg of cream is applied to 2 cm2 of skin (125 mg/kg imiquimod-5% W/V, and/or 625 mg/kg isostearic acid-25% W/V). Comparing cream formulations containing isostearic acid, imiquimod and the combination, we observed that isostearic acid causes skin inflammation within 2 days, whereas imiquimod requires up to 5 days for initial signs. Isostearic acid activated an inflammasome response, stimulated release of proinflammatory cytokines and upregulated the IL-23/17 axis. Animals treated with isostearic acid had enlarged livers (+ 40% weight), which was not observed with imiquimod alone. Imiquimod was readily metabolized and cleared from plasma and liver, but was maintained at high levels in the skin throughout the body (200 mM at area of application; 200 µM in untreated skin). Imiquimod application was associated with splenomegaly, cytokine induction/release and initial body weight loss over 3 days. Despite high imiquimod skin levels throughout the animal, inflammation was only apparent in the treated areas and was less severe than in isostearic acid groups. As the concentrations in these areas are well above the 10 µM required for TLR7 responses in vitro, there is an implication that skin inflammation following imiquimod is due to effects other than TLR7 agonism (e.g., adenosine receptor agonism). In brain, isostearic caused no major changes in cytokine expression while imiquimod alone sightly stimulated expression of IL-1ß and CCL9. However, the combination of both caused brain induction of CCL3, -9, CXCL10, -13, IL-1ß and TNFα. The implication of these data is that isostearic acid facilitates the entry of imiquimod or peripherally secreted cytokines into the brain. Our data suggest that psoriaform skin responses in mice are more driven by isostearic acid, than generally reported and that the dose and route used in the model, leads to profound systemic effects, which may complicate the interpretation of drug effects in this model.

Immune cell targeted fumaric esters support a role of GPR109A as a primary target of monomethyl fumarate in vivo.

Dimethyl fumarate (DMF) is approved as a treatment for multiple sclerosis (MS), however, its mode of action remains unclear. One hypothesis proposes that Michael addition to thiols by DMF, notably glutathione is immunomodulatory. The alternative proposes that monomethyl fumarate (MMF), the hydrolysis product of DMF, is a ligand to the fatty acid receptor GPR109A found in the lysosomes of immune cells. We prepared esters of MMF and macrolides derived from azithromycin, which were tropic to immune cells by virtue of lysosomal trapping. We tested the effects of these substances in an assay of response to Lipopolysaccharide (LPS) in freshly isolated human peripheral blood mononuclear cells (PBMCs). In this system, we observed that the 4'' ester of MMF (compound 2 and 3) reduced levels of Interleukins (IL)-1ß, IL-12 and tumor necrosis factor alpha (TNFα) significantly at a concentration of 1 µM, while DMF required about 25 µM for the same effect. The 2' esters of MMF (compound 1 and 2) were, like MMF itself, inactive in vitro. The 4'' ester formed glutathione conjugates rapidly while the 2' conjugates did not react with thiols but did hydrolyze slowly to release MMF in these cells. We then tested the substances in vivo using the imiquimod/isostearate model of psoriasis where the 2' ester was the most active at 0.06-0.12 mg/kg (approximately 0.1 µmol/kg), improving skin score, body weight and cytokine levels (TNFα, IL-17A, IL-17F, IL-6, IL-1ß, NLRP3 and IL-23A). In contrast, the thiol reactive 4'' ester was less active than the 2' ester while DMF was ca. 300-fold less active. The thiol reactive 4'' ester was not easily recovered from either plasma or organs while the 2' ester exhibited conventional uptake and elimination. The 2' ester also reduced levels of IL-6 in acute monosodium urate (MSU) induced inflammation. These data suggest that mechanisms that are relevant in vivo center on the release of MMF. Given that GPR109A is localized to the lysosome, and that lysosomal trapping increases 2' ester activity by > 300 fold, these data suggest that GPR109A may be the main target in vivo. In contrast, the effects associated with glutathione (GSH) conjugation in vitro are unlikely to be as effective in vivo due to the much lower dose in use which cannot titrate the more concentrated thiols. These data support the case for GPR109A modulation in autoimmune diseases.

The Azithromycin Pro-Drug CSY5669 Boosts Bacterial Killing While Attenuating Lung Inflammation Associated with Pneumonia Caused by Methicillin-Resistant Staphylococcus aureus.

Antibiotic resistance is a major problem, with methicillin-resistant Staphylococcus aureus (MRSA) being a prototypical example in surgical and community-acquired infections. S. aureus, like many pathogens, is immune evasive and able to multiply within host immune cells. Consequently, compounds that aid host immunity (e.g., by stimulating the host-mediated killing of pathogens) are appealing alternatives or adjuncts to classical antibiotics. Azithromycin is both an antibacterial and an immunomodulatory drug that accumulates in immune cells. We set out to improve the immunomodulatory properties of azithromycin by coupling the immune activators, nitric oxide and acetate, to its core structure. This new compound, designated CSY5669, enhanced the intracellular killing of MRSA by 45% ± 20% in monocyte-derived macrophages and by 55% ± 15% in peripheral blood leukocytes, compared with untreated controls. CSY5669-treated peripheral blood leukocytes produced fewer proinflammatory cytokines, while in both monocyte-derived macrophages and peripheral blood leukocytes, phagocytosis, ROS production, and degranulation were unaffected. In mice with MRSA pneumonia, CSY5669 treatment reduced inflammation, lung pathology and vascular leakage with doses as low as 0.01 µmol/kg p.o. CSY5669 had diminished direct in vitro antibacterial properties compared with azithromycin. Also, CSY5669 was immunomodulatory at concentrations well below 1% of the minimum inhibitory concentration, which would minimize selection for macrolide-resistant bacteria if it were to be used as a host-directed therapy. This study highlights the potential of CSY5669 as a possible adjunctive therapy in pneumonia caused by MRSA, as CSY5669 could enhance bacterial eradication while simultaneously limiting inflammation-associated pathology.

The FXR agonist PX20606 ameliorates portal hypertension by targeting vascular remodelling and sinusoidal dysfunction.

BACKGROUND & AIMS: Steroidal farnesoid X receptor (FXR) agonists demonstrated potent anti-fibrotic activities and lowered portal hypertension in experimental models. The impact of the novel non-steroidal and selective FXR agonist PX20606 on portal hypertension and fibrosis was explored in this study. METHODS: In experimental models of non-cirrhotic (partial portal vein ligation, PPVL, 7days) and cirrhotic (carbon tetrachloride, CCl4, 14weeks) portal hypertension, PX20606 (PX,10mg/kg) or the steroidal FXR agonist obeticholic acid (OCA,10mg/kg) were gavaged. We then measured portal pressure, intrahepatic vascular resistance, liver fibrosis and bacterial translocation. RESULTS: PX decreased portal pressure in non-cirrhotic PPVL (12.6±1.7 vs. 10.4±1.1mmHg; p=0.020) and cirrhotic CCl4 (15.2±0.5 vs. 11.8±0.4mmHg; p=0.001) rats. In PPVL animals, we observed less bacterial translocation (-36%; p=0.041), a decrease in lipopolysaccharide binding protein (-30%; p=0.024) and splanchnic tumour necrosis factor α levels (-39%; p=0.044) after PX treatment. In CCl4 rats, PX decreased fibrotic Sirius Red area (-43%; p=0.005), hepatic hydroxyproline (-66%; p<0.001), and expression of profibrogenic proteins (Col1a1, α smooth muscle actin, transforming growth factor ß). CCl4-PX rats had significantly lower transaminase levels and reduced hepatic macrophage infiltration. Moreover, PX induced sinusoidal vasodilation (upregulation of cystathionase, dimethylaminohydrolase (DDAH)1, endothelial nitric oxide synthase (eNOS), GTP-cyclohydrolase1) and reduced intrahepatic vasoconstriction (downregulation of endothelin-1, p-Moesin). In cirrhosis, PX improved endothelial dysfunction (decreased von-Willebrand factor) and normalized overexpression of vascular endothelial growth factor, platelet-derived growth factor and angiopoietins. While short-term 3-day PX treatment reduced portal pressure (-14%; p=0.041) by restoring endothelial function, 14week PX therapy additionally inhibited sinusoidal remodelling and decreased portal pressure to a greater extent (-22%; p=0.001). In human liver sinusoidal endothelial cells, PX increased eNOS and DDAH expression. CONCLUSIONS: The non-steroidal FXR agonist PX20606 ameliorates portal hypertension by reducing liver fibrosis, vascular remodelling and sinusoidal dysfunction. LAY SUMMARY: The novel drug PX20606 activates the bile acid receptor FXR and shows beneficial effects in experimental liver cirrhosis: In the liver, it reduces scarring and inflammation, and also widens blood vessels. Thus, PX20606 leads to an improved blood flow through the liver and decreases hypertension of the portal vein. Additionally, PX20606 improves the altered intestinal barrier and decreases bacterial migration from the gut.

Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models.

Using a novel drug discovery technology reported in previous issues of this journal cyclic peptides have been created which are able to down-regulate secretion of inflammatory cytokines, in vitro, by stimulated cells of the macrophage cell line J774. The cytokines in question, TNF-alpha and IL-6, are strongly implicated in etiology of diseases such as rheumatoid arthritis. Studies are reported here using the CAIA animal model for rheumatoid arthritis, which show that the peptides identified are indeed able to impact on inflammation of joints, induced in vivo. The results suggest that these peptides are effective at a dose which could be viable in man, and at which no adverse side effects are evident in the short term.

Novel substituted isoxazole FXR agonists with cyclopropyl, hydroxycyclobutyl and hydroxyazetidinyl linkers: Understanding and improving key determinants of pharmacological properties.

Several isoxazole-containing series of FXR agonists have been published over the last 15years, subsequent to the prototypical amphiphilic 'hammerhead'-type structure that was originally laid out by GW4064, the first potent synthetic FXR agonist. A set of novel compounds where the hammerhead is connected to the terminal carboxylic acid-bearing aryl or heteroaryl moiety by either a cyclopropyl, a hydroxycyclobutyl or a hydroxyazetidinyl linker was synthesized in order to improve upon the ADME properties of such isoxazoles. The resulting compounds all demonstrated high potencies at the target receptor FXR but with considerable differences in their physicochemical and in vivo profiles. The structure-activity relationships for key chemical features that have a major impact on the in vivo pharmacology of this series are discussed.

The nuclear bile acid receptor FXR controls the liver derived tumor suppressor histidine-rich glycoprotein.

The nuclear bile acid receptor Farnesoid X receptor (FXR) is strongly expressed in liver and intestine, controls bile acid and lipid homeostasis and exerts tumor-protective functions in liver and intestine. Histidine-rich glycoprotein (HRG) is an abundant plasma protein produced by the liver with the proposed function as a pattern recognition molecule involved in the clearance of immune complexes, necrotic cells and pathogens, the modulation of angiogenesis, the normalization of deranged endothelial vessel structure in tumors and tumor suppression. FXR recognition sequences were identified within a human HRG promoter fragment that mediated FXR/FXR-agonist dependent reporter gene activity in vitro. We show that HRG is a novel transcriptional target gene of FXR in human hepatoma cells, human upcyte® primary hepatocytes and 3D human liver microtissues in vitro and in mouse liver in vivo. Prolonged administration of the potent nonsteroidal FXR agonist PX20606 increases HRG levels in mouse plasma. Finally, daily oral administration of this FXR agonist for seven days resulted in a significant increase of HRG levels in the plasma of healthy human male volunteers during a clinical Phase I safety study. HRG might serve as a surrogate marker indicative of liver-specific FXR activation in future human clinical studies. Furthermore, potent FXR agonists might be beneficial in serious health conditions where HRG is reduced, for example, in hepatocellular carcinoma but also other solid cancers, liver failure, sepsis and pre-eclampsia.

Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio.

Janus kinase (JAK) inhibitors act at low doses (e.g., tofacitinib, 0.2-0.4 µmol/kg bid) in clinical use, suggesting an efficient underlying mode of action. We hypothesized that their effectiveness is due to their ability to raise the ratio of IL-10 to TNFα. Unlike other JAK isoforms, JAK3 is expressed mainly in hematopoietic cells and is essential for immune function. We used JAK3 selective inhibitors with preferential distribution to immune cells. Inhibition of JAK3 in human leukocytes reduced TNFα and IL-6 but maintained levels of IL-10, while pan-JAK inhibitors increased TNFα, IL-6, and IL-10. JAK1 is required for IL-10 receptor signaling, which suggests that, at exposure above the IC50 (55 nM for tofacitinib on JAK1), there is less feedback control of TNFα levels. This leads to self-limiting effects of JAK1 inhibitors and could place an upper limit on appropriate doses. In vivo, treating mice with JAK3 inhibitors before LPS administration decreased plasma TNFα and increased IL-10 above vehicle levels, suggesting that JAK3 inhibition may limit TNFα release by increasing IL-10 while leaving the IL-10 receptor functional. This mechanism should have general utility in controlling autoimmune diseases and can be conveniently observed by measuring the ratio of IL-10 to TNFα. In summary, our targeted, "leukotropic" inhibitors more effectively increased IL-10/TNFα ratios than unselective control compounds and could, therefore, be ideal for autoimmune therapy.

Inherent Fluorescence Demonstrates Immunotropic Properties for Novel Janus Kinase 3 Inhibitors.

There is a general question in small molecule pharmacology about how apparent compound concentrations in blood, plasma, and organs actually relate to actual amounts at the target site of a compound. In this study, we used inherently fluorescent JAK3 ligands and their macrolide conjugates to investigate the relationship between physical properties, apparent bulk concentration, and organ and subcellular distribution. In vitro uptake into immune cells suggested that much of the substance was associated with granules or organelles. Samples from murine pharmacokinetic studies were analyzed by both conventional mass spectrometry and cryofluorescence microscopy methods to show the distribution of a compound within organs and cells without artifacts of fixation. These observations confirm the uptake of granules observed in vitro. Data from macrolides carrying either a coumarin fluorophore or a JAK3 inhibitor were similar, suggesting that the distribution is directed by the properties of the larger macrolide. These data show a propensity for azalide macrolides to concentrate in the lung and gut epithelia and suggest that the plasma- or whole-blood-derived estimates of drug levels almost certainly underestimate concentrations of macrolides in the mucous membranes. Thus, their apparent efficacy at sub-bacteriostatic doses may reflect their higher levels in barrier layers.

Imidazoquinolines with improved pharmacokinetic properties induce a high IFNα to TNFα ratio in vitro and in vivo.

TLR Agonists have promising activity in preclinical models of viral infection and cancer. However, clinical use is only in topical application. Systemic uses of TLR-ligands such as Resiquimod, have failed due to adverse effects that limited dose and thus, efficacy. This issue could be related to pharmacokinetic properties that include fast elimination leading to low AUC with simultaneously high cmax at relevant doses. The high cmax is associated with a sharp, poorly tolerated cytokine pulse, suggesting that a compound with a higher AUC/cmax-ratio could provide a more sustained and tolerable immune activation. Our approach was to design TLR7/8-agonist Imidazoquinolines intended to partition to endosomes via acid trapping using a macrolide-carrier. This can potentially extend pharmacokinetics and simultaneously direct the compounds to the target compartment. The compounds have hTLR7/8-agonist activity (EC50 of the most active compound in cellular assays: 75-120 nM hTLR7, 2.8-3.1 µM hTLR8) and maximal hTLR7 activation between 40 and 80% of Resiquimod. The lead candidates induce secretion of IFNα from human Leukocytes in the same range as Resiquimod but induce at least 10-fold less TNFα in this system, consistent with a higher specificity for human TLR7. This pattern was reproduced in vivo in a murine system, where small molecules are thought not to activate TLR8. We found that Imidazoquinolines conjugated to a macrolide or, substances carrying an unlinked terminal secondary amine, had longer exposure compared with Resiquimod. The kinetics of pro-inflammatory cytokine release for these substances in vivo were slower and more extended (for comparable AUCs, approximately half-maximal plasma concentrations). Maximal IFNα plasma levels were reached 4 h post application. Resiquimod-treated groups had by then returned to baseline from a peak at 1 h. We propose that the characteristic cytokine profile is likely a consequence of altered pharmacokinetics and, potentially, enhanced endosomal tropism of the novel substances. In particular, our substances are designed to partition to cellular compartments where the target receptor and a distinct combination of signaling molecules relevant to IFNα-release are located. These properties could address the tolerability issues of TLR7/8 ligands and provide insight into approaches to fine-tune the outcomes of TLR7/8 activation by small molecules.

Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (-/-) mice.

Farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, plays an important role in the regulation of cholesterol and more specifically high-density lipoprotein (HDL) homeostasis. Activation of FXR is reported to lead to both pro- and anti-atherosclerotic effects. In the present study we analyzed the impact of different FXR agonists on cholesterol homeostasis, plasma lipoprotein profiles, and transhepatic cholesterol efflux in C57BL/6J mice and cynomolgus monkeys and atherosclerosis development in cholesteryl ester transfer protein transgenic (CETPtg) low-density lipoprotein receptor (LDLR) (-/-) mice. In C57BL/6J mice on a high-fat diet the synthetic FXR agonists isopropyl 3-(3,4-difluorobenzoyl)-1,1-dimethyl-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (FXR-450) and 4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl]benzoic acid (PX20606) demonstrated potent plasma cholesterol-lowering activity that affected all lipoprotein species, whereas 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) and 6-ethyl chenodeoxycholic acid (6-ECDCA) showed only limited effects. In FXR wild-type mice, but not FXR(-/-) mice, the more efficacious FXR agonists increased fecal cholesterol excretion and reduced intestinal cholesterol (re)uptake. In CETPtg-LDLR(-/-) mice PX20606 potently lowered total cholesterol and, despite the observed HDL cholesterol (HDLc) reduction, caused a highly significant decrease in atherosclerotic plaque size. In normolipidemic cynomolgus monkeys PX20606 and 6-ECDCA both reduced total cholesterol, and PX20606 specifically lowered HDL(2c) but not HDL(3c) or apolipoprotein A1. That pharmacological FXR activation specifically affects this cholesterol-rich HDL(2) subclass is a new and highly interesting finding and sheds new light on FXR-dependent HDLc lowering, which has been perceived as a major limitation for the clinical development of FXR agonists.

Pharmacokinetics, Pharmacodynamics and Antiviral Efficacy of the MEK Inhibitor Zapnometinib in Animal Models and in Humans.

The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib (12.5 mg/kg p. o. twice daily, 8 h interval). Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (Cmax) was reached between 2-4 h after administration with an elimination half-life of 4-5 h in dogs, 8 h in mice or hamsters and 19 h in human subjects. Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 µg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%-80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 µg/ml zapnometinib in further clinical studies.

Folate-Targeted Liposomal Formulations Improve Effects of Methotrexate in Murine Collagen-Induced Arthritis.

Methotrexate (MTX) is first-line therapy for the treatment of rheumatoid arthritis (RA), however, its use may be limited by side effects notably post-injection malaise. When patients are intolerant or become unresponsive, second-line or antibody therapy may be indicated. A folate-targeted liposomal formulation of MTX (FL-MTX) is tropic to arthritic paws and prevents the onset of collagen-induced arthritis (CIA) in the mouse. We optimized the drug-to-lipid molar ratio to 0.15 and demonstrated the therapeutic efficacy of this form at 2 mg/kg MTX intraperitoneal (i.p.) twice a week. These improved liposomes were present in inflamed joints in proportion to the degree of swelling of the paw and bone remodeling activity. FL-MTX had lower hepatic and renal elimination of MTX than the free substance. FL-MTX provided equivalent results when given i.p. or subcutaneous (s.c.) and FL-MTX 2 mg/kg (drug/lipid 0.15), twice weekly, was similar to or more effective than 35 mg/kg MTX (same route and schedule) in reducing the incidence and swelling in the murine CIA model. These results suggest that FL-MTX is a more potent nanotherapeutic formulation than free MTX treatment. Its potential benefits for patients may include reduced frequency of treatment and lower overall doses for a given response.

Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors.

Stress-induced p38α mitogen-activated protein (MAP) kinase activation modulates cytokine overproduction and is associated with neuroinflammation and neurodegeneration. As a potential therapeutic approach, novel Skepinone-based p38α MAP kinase inhibitors were optimized to cross the blood-brain barrier via either amino acid transporters or hydrophobic diffusion. To enhance absorption from the oral route, we used methyl ester prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (43; p38α, IC50 = 5.5 nM) and 4-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC50 = 12 nM) had brain-to-plasma ratios of 1.4 and 4.4, respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (p38α, IC50 = 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

A Host-Directed Approach to the Detection of Infection in Hard-to-Heal Wounds.

Wound infection is traditionally defined primarily by visual clinical signs, and secondarily by microbiological analysis of wound samples. However, these approaches have serious limitations in determining wound infection status, particularly in early phases or complex, chronic, hard-to-heal wounds. Early or predictive patient-derived biomarkers of wound infection would enable more timely and appropriate intervention. The observation that immune activation is one of the earliest responses to pathogen activity suggests that immune markers may indicate wound infection earlier and more reliably than by investigating potential pathogens themselves. One of the earliest immune responses is that of the innate immune cells (neutrophils) that are recruited to sites of infection by signals associated with cell damage. During acute infection, the neutrophils produce oxygen radicals and enzymes that either directly or indirectly destroy invading pathogens. These granular enzymes vary with cell type but include elastase, myeloperoxidase, lysozyme, and cathepsin G. Various clinical studies have demonstrated that collectively, these enzymes, are sensitive and reliable markers of both early-onset phases and established infections. The detection of innate immune cell enzymes in hard-to-heal wounds at point of care offers a new, simple, and effective approach to determining wound infection status and may offer significant advantages over uncertainties associated with clinical judgement, and the questionable value of wound microbiology. Additionally, by facilitating the detection of early wound infection, prompt, local wound hygiene interventions will likely enhance infection resolution and wound healing, reduce the requirement for systemic antibiotic therapy, and support antimicrobial stewardship initiatives in wound care.

Removal of RPE lipofuscin results in rescue from retinal degeneration in a mouse model of advanced Stargardt disease: Role of reactive oxygen species.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging and is associated with retinal degeneration encountered in age-related macular degeneration (AMD) and Stargardt disease (SD). Currently, treatment for lipofuscin-induced retinal degeneration is unavailable. Here, we report that Remofuscin (INN: soraprazan, a tetrahydropyridoether small molecule) reverses lipofuscin accumulation in aged primary human RPE cells and is non-cytotoxic in aged SD mouse RPE cells in vitro. In addition, we show that the removal of lipofuscin after a single intravitreal injection of Remofuscin results in a rescue from retinal degeneration in a mouse model of advanced SD which is even accompanied by an amelioration of the retinal dysfunction. Finally, we demonstrate that the mechanism causing lipofuscinolysis may involve the reactive oxygen species generated via the presence of Remofuscin. These data suggest a possible therapeutic approach to untreatable lipofuscin-mediated diseases like AMD, SD and lipofuscinopathies in neurodegenerative diseases.

Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells.

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

A potassium channel agonist protects hearing function and promotes outer hair cell survival in a mouse model for age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory impairment mainly caused by degeneration of sensory hair cells in the cochlea with no causal medical treatment available. Auditory function and sensory hair cell survival critically depend on the Kv7.4 (KCNQ4) channel, a voltage-gated potassium channel expressed in outer hair cells (OHCs), with its impaired function or reduced activity previously associated with ARHL. Here, we investigated the effect of a potent small-molecule Kv7.4 agonist on ARHL in the senescence-accelerated mouse prone 8 (SAMP8) model. For the first time in vivo, we show that Kv7.4 activation can significantly reduce age-related threshold shifts of auditory brainstem responses as well as OHC loss in the SAMP8 model. Pharmacological activation of Kv7.4 thus holds great potential as a therapeutic approach for ARHL as well as other hearing impairments related to Kv7.4 function.

Synthesis, Characterization, and in†vivo Distribution of Intracellular Delivered Macrolide Short-Chain Fatty Acid Derivatives.

Short-chain fatty acids (SCFAs) have a range of effects in metabolism and immune regulation. We have observed that delivery of SCFAs to lysosomes has potent immune regulatory effects, possibly as a surrogate signal for the presence of anaerobic organisms. To better understand the pharmacology of lysosomal SCFA donors, we investigated the distribution and metabolism of propionate and butyrate donors. Each analog (1 a and 2 a) can donate three SCFA equivalents via ester hydrolysis through six intermediate metabolites. The compounds are stabilized by low pH, and stability in cells is usually higher than in medium, but is cell-type specific. Butyrate derivatives were found to be more stable than propionates. Tri-esters were more stable than di- or mono-esters. The donors were surprisingly stable in vivo, and hydrolysis of each position was organ specific. Jejunum and liver caused rapid loss of 4'' esters. The gut metabolite pattern by i. v. differed from that of p.o. application, suggesting luminal and apical enzyme effects in the gut epithelium. Central organs could de-esterify the 11-position. Levels in lung relative to other organs were higher by p.o. than via i. v., suggesting that delivery route can influence the observed pharmacology and that gut metabolites distribute differently. The donors were largely eliminated by 24 h, following near linear decline in organs. The observed levels and distribution were found to be consistent with pharmacodynamic effects, particularly in the gut.

Complementary methods for SARS-CoV-2 diagnosis in times of material shortage.

The pandemic caused by SARS-CoV-2 resulted in increasing demands for diagnostic tests, leading to a shortage of recommended testing materials and reagents. This study reports on the performance of self-sampled alternative swabbing material (ordinary Q-tips tested against flocked swab and rayon swab), of reagents for classical RNA extraction (phenol/guanidine-based protocol against a commercial kit), and of intercalating dye-based one-step quantitative reverse transcription real-time PCRs (RT-qPCR) compared against the gold standard hydrolysis probe-based assays for SARS-CoV-2 detection. The study found sampling with Q-tips, RNA extraction with classical protocol and intercalating dye-based RT-qPCR as a reliable and comparably sensitive strategy for detection of SARS-CoV-2-particularly valuable in the current period with a resurgent and dramatic increase in SARS-CoV-2 infections and growing shortage of diagnostic materials especially for regions limited in resources.