Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. ...Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRAS
PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF-PDE8A protein-protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF-cAMP/PKA signaling axes and treating KRAS-c-RAF dependent PDAC. Through disrupting the c-RAF-PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRAS
PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS-c-RAF dependent PDAC.
Background and purpose:
A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB
1
and CB
2
receptors. We have shown ...previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB
1
receptors expressed in mouse brain and to human CB
2
receptors that have been transfected into CHO cells.
Experimental approach:
The
35
SGTP
γ
S binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and
R
‐(+)‐WIN55212) at the mouse CB
1
and the human CB
2
receptor.
Key results:
This paper reports firstly that cannabidiol displays inverse agonism at the human CB
2
receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB
2
receptors.
Conclusions and implications:
This study has provided the first evidence that cannabidiol can display CB
2
receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB
2
receptor. The ability of cannabidiol to behave as a CB
2
receptor inverse agonist may contribute to its documented anti‐inflammatory properties.
British Journal of Pharmacology
(2007)
150
, 613–623. doi:
10.1038/sj.bjp.0707133
The issue of chemically reactive drug metabolites is one of growing concern in the pharmaceutical industry inasmuch as some, but not all, reactive intermediates are believed to play a role as ...mediators of drug-induced toxicities. While it is now relatively straightforward to identify these short-lived electrophilic species through appropriate in vitro “trapping” experiments, our current understanding of mechanistic aspects of xenobiotic-induced toxicities is such that we cannot predict which reactive intermediates are likely to cause a toxic insult and which will be benign. Little is known about the identities of the macromolecular targets (primarily proteins) of these electrophiles or the functional consequences of their covalent modification by reactive drug metabolites. As a result, several companies have adopted approaches to minimize the potential for metabolic activation of drug candidates at the discovery/lead optimization phase as a default strategy. However, research leading to a deeper insight into mechanistic aspects of toxicities caused by reactive drug metabolites will aid greatly in the rational design of drug candidates with superior safety profiles and represents a challenging and exciting opportunity for chemical toxicology.
It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug−protein adducts and organ toxicity. Because the potential does exist, however, for ...these processes to be causally related, the general strategy at Merck Research Laboratories has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metabolism vectors and typically involves the initial use of small molecule trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) preparations from preclinical species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Laboratories. The availability of a dedicated Labeled Compound Synthesis group, coupled to a close working relationship between Drug Metabolism and Medicinal Chemistry, represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.
The recommendations of the FDA’s final Guidance document on Safety Testing of Drug Metabolites provide a framework for devising preclinical toxicology assessment paradigms, where necessary, for human ...metabolites of small molecule drug candidates. Importantly, these recommendations carry implications for the qualitative and quantitative analysis of circulating drug metabolites in early human trials, which typically are performed without the benefit of a radiolabeled tracer. In this perspective, an approach to these goals is outlined based on recent work at Merck Research Laboratories involving the use of ultraperformance liquid chromatography−mass spectrometry analysis, performed on a high-resolution time-of-flight mass spectrometer, of first-in-human study plasma samples. With the aid of a fractional mass filtering algorithm, drug metabolites are distinguished from endogeneous background materials and subsequently identified on the basis of their accurate masses, product ion mass spectra, and computer-assisted structure elucidation software routines. Semiquantitative analysis then is based on calibration of the MS response to each analyte with reference to radioactivity data from in vitro metabolic profiles. In the case of chemically reactive drug metabolites, which are excluded from consideration in the Guidance, a proactive approach is advocated whereby potent (low dose) drug candidates with only a limited propensity to form electrophilic intermediates are advanced into development. Overall, a decision on the need to conduct separate evaluation of the safety profile of a human drug metabolite(s) should take into consideration all of the available information on the compound of interest and be based on a case-by-case approach employing sound scientific principles.
The human pharmacokinetics, metabolism, and excretion of
C-ganaxolone (GNX) were characterized in healthy male subjects (
= 8) following a single 300-mg (150
Ci) oral dose. GNX exhibited a short ...half-life of 4 hours in plasma, whereas total radioactivity had a half-life of 413 hours indicating extensive metabolism to long-lived metabolites. Identification of the major GNX circulating metabolites required extensive isolation and purification for liquid chromatography-tandem mass spectrometry analysis, together with in vitro studies, NMR spectroscopy, and synthetic chemistry support. This revealed that the major routes of GNX metabolism involved hydroxylation at the 16
-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20
-hydroxysterol, and sulfation of the 3
-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H
SO
to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3
-methyl substituent to a carboxylic acid and sulfation at the 20
position, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identification of no less than 59 metabolites of GNX, demonstrated the high complexity of the metabolic fate of this drug in humans and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. SIGNIFICANCE STATEMENT: Studies on the metabolism of
C-ganaxolone in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step pathway. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of traditional animal studies in predicting major circulating metabolites in man.
Periodontal disease is more prevalent in patients with diabetes, and it has a negative impact on their quality of life. Inhibiting the infection and inflammation processes that cause periodontal ...disease can reduce the severity of the disease and chances of serious complications. In this study, we aimed to demonstrate the effectiveness of Clinacanthus nutans extract in reducing the inflammation in gingival fibroblast cells induced by Porphyromonas gingivalis lipopolysaccharide (LPS). Stimulation with LPS under high-glucose conditions led to increased inflammation compared to low-glucose conditions. Treatment of C. nutans extract significantly reduced the expression of these pro-inflammatory cytokines and chemokines. At a concentration of 50 μg/mL, it reduced the relative expression of IL6, IL8, and CXCL10 to 0.51 ± 0.09, 0.6 ± 0.19, and 0.09 ± 0.02, respectively, compared to the non-treatment control, accompanied by a decrease in secreted protein as measured by ELISA. Additionally, application of C. nutans extract markedly suppressed the NF-κB signaling pathway by reducing the phosphorylated form of IκBα, NF-κB p65, and nuclear translocation of NF-κB, along with a decrease in COX2, a key mediator in the inflammatory pathway. Furthermore, analysis of RNA sequencing data indicated that the extract clearly reversed the gene expression changes induced by LPS. This was particularly true for the signaling mediators and inflammatory genes in response to NF-κB, JAK/STAT, and TNF signaling pathways. Our finding highlights the potential of C. nutans extract to alleviate inflammation and suggests its potential as a treatment for periodontal disease in patients with diabetes.Periodontal disease is more prevalent in patients with diabetes, and it has a negative impact on their quality of life. Inhibiting the infection and inflammation processes that cause periodontal disease can reduce the severity of the disease and chances of serious complications. In this study, we aimed to demonstrate the effectiveness of Clinacanthus nutans extract in reducing the inflammation in gingival fibroblast cells induced by Porphyromonas gingivalis lipopolysaccharide (LPS). Stimulation with LPS under high-glucose conditions led to increased inflammation compared to low-glucose conditions. Treatment of C. nutans extract significantly reduced the expression of these pro-inflammatory cytokines and chemokines. At a concentration of 50 μg/mL, it reduced the relative expression of IL6, IL8, and CXCL10 to 0.51 ± 0.09, 0.6 ± 0.19, and 0.09 ± 0.02, respectively, compared to the non-treatment control, accompanied by a decrease in secreted protein as measured by ELISA. Additionally, application of C. nutans extract markedly suppressed the NF-κB signaling pathway by reducing the phosphorylated form of IκBα, NF-κB p65, and nuclear translocation of NF-κB, along with a decrease in COX2, a key mediator in the inflammatory pathway. Furthermore, analysis of RNA sequencing data indicated that the extract clearly reversed the gene expression changes induced by LPS. This was particularly true for the signaling mediators and inflammatory genes in response to NF-κB, JAK/STAT, and TNF signaling pathways. Our finding highlights the potential of C. nutans extract to alleviate inflammation and suggests its potential as a treatment for periodontal disease in patients with diabetes.
Phosphodiesterase type 4 (PDE4) enzymes specifically hydrolyse cAMP in many cell signalling systems that are transduced by hormones and other primary messengers. The physiological function of the ...four PDE4 subfamilies (A, B, C and D) are numerous and varied due to the differentially localised plethora of isoforms that can be detected in cardiovascular, CNS and immune systems. Of the four subfamilies, least is known about PDE4C probably due to its restricted distribution pattern, scarcity of selective inhibitors and the lack of developed research tools. Here, for the first time, we chart the discovery of PDE4C, describe its regulation and highlight cancers where future development of PDE4C selective small molecules may have potential.
Deutetrabenazine (Austedo, Teva Pharmaceuticals) is a deuterated form of tetrabenazine. It is the first deuterated drug to receive US regulatory approval and is approved for treatment of chorea in ...Huntington’s disease and tardive dyskinesia. Two oral single dose studies comparing deutetrabenazine (25 mg) with tetrabenazine (25 mg) in healthy volunteers evaluated the impact of deuteration on pharmacokinetics of the active metabolites, alpha‐dihydrotetrabenazine (α‐HTBZ) and beta‐dihydrotetrabenazine (β‐HTBZ), metabolite profile, safety, and tolerability. In the two‐way, cross‐over study, the mean elimination half‐life of deuterated total (α + β)‐HTBZ was doubled compared with nondeuterated total (α + β)‐HTBZ, with a twofold increase in overall mean exposure (area under the concentration‐time curve from zero to infinity (AUC0–inf)) and a marginal increase in mean peak plasma concentration (Cmax). In the mass balance and metabolite profiling study, there were no novel plasma or urinary metabolites of 14C‐deutetrabenazine relative to 14C‐tetrabenazine. Specific deuteration in deutetrabenazine resulted in a superior pharmacokinetic profile and an increased ratio of active‐to‐inactive metabolites, attributes considered to provide significant benefits to patients.
Abstract
This article provides a review of Sid Nelson's key contributions to the fields of drug metabolism and toxicology over a long and distinguished career. Selected examples are discussed to ...illustrate the diversity of Sid's research, with an emphasis on understanding mechanistic aspects of metabolic activation processes and structure-toxicity relationships. These examples serve to illustrate the importance of emerging mass spectrometry and isotope labeling techniques in elucidating details of foreign compound metabolism at the molecular level, an area in which Sid pioneered most effectively.
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