Techniques and instrumentation that involve fluorescence are used in many careers related to biology and biochemistry. However, due to the expense of instrumentation, the theory and practice of ...fluorescence spectroscopy is commonly concentrated in instrumental analysis in most curricula, a class rarely required for biology and biochemistry majors. Biochemistry represents a course taken by these majors, providing an opportunity to expose them to the fundamentals of fluorescence. A three session laboratory series has been developed where the kinetic analysis of lactate dehydrogenase is carried out through the use of inexpensive, student-built fluorometers. These instruments are optimized for the detection of NADH fluorescence and constructed with interlocking building blocks (such as LEGO blocks), an LED source, a phototransistor detector, and other commercially available materials for a total cost of $52. Biochemistry students have built these spectrometers and used them to determine the K m of Gallus gallus lactate dehydrogenase for L-lactate and the k cat of this enzyme in accordance with literature values. Learning outcomes were assessed and indicate proficient understanding of instrument use and design.
PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To ...facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed. This formulation was tested in 2 rat ulcerative colitis models and evaluated for distribution,
, in rats, dogs, and humans.
The rat models of colitis were induced by treatment with 2,4-dinitrobenzenesulfonic acid or dextran sulfate sodium. Single nuclei RNA sequencing of colon tissues was performed to characterize the mechanism of action. The distribution and concentration of PL8177 and the main metabolite within the GI tract after a single oral dose of PL8177 was investigated in rats and dogs. A phase 0 clinical study using a single microdose (70 µg) of
C-labeled PL8177 investigated the release of PL8177 in the colon of healthy men after oral administration.
Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. Histopathology analysis resulted in the maintenance of intact colon structure and barrier, reduced immune cell infiltration, and increased enterocytes with PL8177 treatment. Transcriptome data show that oral PL8177 50 µg treatment causes relative cell populations and key gene expressions levels to move closer to healthy controls. Compared with vehicle, treated colon samples show negative enrichment of immune marker genes and diverse immune-related pathways. In rats and dogs, orally administered PL8177 was detected at higher amounts in the colon vs upper GI tract.
C-PL8177 and the main metabolite were detected in the feces but not in the plasma and urine in humans. This suggests that the parent drug
C-PL8177 was released from the polymer formulation and metabolized within the GI tract, where it would be expected to exert its effect.
Collectively, these findings support further research into the oral formulation of PL8177 as a possible therapeutic for GI inflammatory diseases in humans.
Background
Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC
1
) as reported in several ...experimental settings. As such, MC
1
can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC
1
agonist (PL8177)
in vitro
and in a mouse model of inflammatory arthritis.
Methods
PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC
1,3,4,5
), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the
in vivo
potential of PL8177.
Results
PL8177 activates mouse and human MC
1
with apparent EC
50
values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC
50
: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint.
Conclusion
These results demonstrate that the MC
1
agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis.
Significance
Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC
1
), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated
in vivo
efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC
1
receptor, as a promising strategy to manage chronic inflammatory diseases.
The heterogeneity and anisotropy of fractured-rock aquifers, such as those in the Columbia River Basalt Province, present challenges for determining groundwater recharge. The entrance of recharge to ...the fractured-basalt and interbedded-sediment aquifer in the Palouse region of north-central Idaho is not well understood because of successive basalt flows that act as restrictive barriers. It was hypothesized that a primary recharge zone exists along the basin’s eastern margin at a mountain-front interface where eroded sediments form a more conductive zone for recharge. Potential source waters and groundwater were analyzed for δ18O and δ2H to discriminate recharge sources and pathways. Snowpack values ranged from −22 to −12‰ for δ18O and from −160 to −90‰ for δ2H and produced spring-time snowmelt ranging from −16.5 to −12‰ for δ18O and from −120 to −90‰ for δ2H. With the transition of snowmelt to spring-time ephemeral creeks, the isotope values compressed to −16 and −14‰ for δ18O and −110 and −105‰ for δ2H. A greater range of values was present for a perennial creek (−18 to −13.5‰ for δ18O and −125 to −98‰ for δ2H) and groundwater (−17.5 to −13‰ for δ18O and −132 to −105‰ for δ2H), which reflect a mixing of seasonal signals and the varying influence of vapor sources and sublimation/evaporation. Inverse modeling and the evaluation of matrix characteristics indicate conductive pathways associated with paleochannels and deeper pathways along the mountain-front interface. Depleted isotope signals indicate quicker infiltration and recharge pathways that were separate from, or had limited mixing with, more evaporated water that infiltrated after greater time/travel at the surface.
The melanocortin α-melanocyte stimulating hormone (α-MSH), an endogenous peptide with high affinity for the melanocortin 1 receptor (MC1r), has demonstrated prevention and reversal of intestinal and ...ocular inflammation in animal models. Preclinical studies were performed to determine whether two MC1r receptor agonists, PL-8177 and PL-8331, exhibit actions and efficacy similar to α-MSH in preventing and reversing intestinal and ocular inflammation.
Both PL-8177 and PL-8331 were assessed in a Eurofins LeadProfilingScreen selectivity panel including 72
assays. PL-8177 and PL-8331 were evaluated in an
assay using human whole blood stimulated by lipopolysaccharide to determine inhibition of tumor necrosis factor alpha (TNF-α); for comparison, adrenocorticotropic hormone (ACTH) and α-MSH were used as positive controls. PL-8177, dosed at 0.5, 1.5, and 5.0 μg, was assessed in a cannulated rat model of dinitrobenzene sulfonic acid (DNBS)-induced bowel inflammation versus vehicle and oral sulfasalazine. PL-8177 was also dosed at 0.3 mg/kg/mouse injected intraperitoneally versus untreated controls and α-MSH treatment in mice with experimental autoimmune uveitis (EAU). PL-8331 at 3 doses, 3 times daily, was evaluated in a murine model of scopolamine-induced dry eye disease (SiccaSystem
model), versus twice-daily Restasis
and Xiidra
.
Both PL-8177 and PL-8331 demonstrated no significant activity at the 1 μm concentration in any of the 72
assays. PL-8177 and PL-8331 inhibited lipopolysaccharide-induced TNF-α to a similar degree as ACTH and α-MSH. In the DNBS rat model of bowel inflammation, PL-8177 was significantly superior to untreated controls at all 3 doses (
< 0.05) in reducing bowel inflammation parameters, with effects similar to sulfasalazine. In the murine EAU model, PL-8177 significantly reduced retinal inflammation scores versus untreated controls (
= 0.0001) over 3-5 weeks, and to a similar degree as α-MSH. In the murine scopolamine-induced model of dry eye disease, PL-8331 reduced corneal fluorescein staining scores at all doses, significantly (
= 0.02) for the highest dose (1 × 10
mg⋅mL
), and similarly to Restasis
; Xiidra
demonstrated no effect.
The MC1r receptor agonists PL-8177 and PL-8331 exhibited actions similar to those of α-MSH in preventing and reversing intestinal and ocular inflammation in preclinical disease models.
Structurally novel 5H-chromeno2,3-bpyridine (azaxanthene) selective glucocorticoid receptor (GR) modulators have been identified. A screening paradigm utilizing cellular assays of GR-mediated ...transrepression of proinflammatory transcription factors and transactivation of GR-dependent genes combined with three physiologically relevant assays of cytokine induction in human whole blood has allowed for the identification of high affinity, selective GR ligands that display a broad range of pharmacological profiles. Agonist efficacy in reporter assays can be tuned by halogenation of a pendent phenyl ring and correlates well with efficacy for cytokine inhibition in human whole blood. A hypothetical binding mode is proposed, invoking an expanded ligand binding pocket resembling that of arylpyrazole-bound GR structures. Two compounds of close structural similarity (35 and 37; BMS-776532 and BMS-791826, respectively) have been found to maintain distinct and consistent levels of partial agonist efficacy across several assays, displaying anti-inflammatory activity comparable to that of prednisolone 2 in suppressing cytokine production in whole blood and in rodent models of acute and chronic inflammation.
The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-α and IL-1β. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as ...rheumatoid arthritis and Crohn's disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-α in vivo.
A novel series of imidazopyrimidines have been developed that potently inhibit p38α and suppress the production of TNF-α in vivo.
The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is ...described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp2 character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.
In search for prodrugs to address the issue of pH-dependent solubility and exposure associated with 1 (BMS-582949), a previously disclosed phase II clinical p38α MAP kinase inhibitor, a structurally ...novel clinical prodrug, 2 (BMS-751324), featuring a carbamoylmethylene linked promoiety containing hydroxyphenyl acetic acid (HPA) derived ester and phosphate functionalities, was identified. Prodrug 2 was not only stable but also water-soluble under both acidic and neutral conditions. It was effectively bioconverted into parent drug 1 in vivo by alkaline phosphatase and esterase in a stepwise manner, providing higher exposure of 1 compared to its direct administration, especially within higher dose ranges. In a rat LPS-induced TNFα pharmacodynamic model and a rat adjuvant arthritis model, 2 demonstrated similar efficacy to 1. Most importantly, it was shown in clinical studies that prodrug 2 was indeed effective in addressing the pH-dependent absorption issue associated with 1.
PDF
