New research conducted by the University of Liverpool and AKL Research and Development Ltd (AKLRD), published in Inflammopharmacology, highlights the potential benefits of a new drug treatment on the human body’s immune response in inflammation.
In a number of inflammatory conditions, such as osteoarthritis, rheumatoid arthritis and, more recently, COVID-19, major complications and extensive tissue damage can occur when the immune system becomes excessively and uncontrollably activated.
Finding new ways to selectively control this over-activity could have major clinical benefits.
Neutrophils
To be healthy, we need an effective immune response, otherwise we would succumb to overwhelming infection, even by everyday bacteria. However, sometimes our immune system can become hyperactive and cause damage through inflammation, even in the absence of any infection.
This can sometimes be extreme. Indeed, many rheumatic diseases such as rheumatoid arthritis and osteoarthritis are caused by inflammation. The quest has always been to find ways to selectively block the harmful effects of an overactive immune system, without paying the price of blocking our ability to fight infections.
Neutrophils are the most abundant immune cells in our blood. They are rapidly dispatched to sites of infection, where they fulfil their life-saving antimicrobial functions by destroying infectious organisms and producing signalling proteins called cytokines, that help co-ordinate the recruitment and activity of other immune system cells to the battle against the infection.
There is much evidence from work in Liverpool to show that these cells are important players behind many rheumatic diseases
In some situations, if the levels of cytokines are too high, they can trigger an extreme inflammatory reaction called a cytokine storm. These storms cause overwhelming inflammation that leads to blocked or ruptured blood vessels.
This can affect the entire circulatory system. Cytokine storms can cause immense damage, multiple organ failure, sepsis, and even death and, appear to play a major role in severe COVID-19 disease.
For many decades scientists and clinicians have understood the potential benefit of suppressing neutrophils, but any attempt to do this without weakening the immune response to infection has failed.
APPA
APPA is a novel drug under development by AKLRD for use in osteoarthritis, a major disabling problem world-wide that is caused by low grade inflammation. The first part of its formal clinical evaluation in Liverpool, led by rheumatologist Professor Robert Moots, has recently been successfully completed.
Now, in partnership between Liverpool and AKLRD, the impact of the drug on neutrophils has been examined and published.
The study found that APPA clearly demonstrated anti-inflammatory potential but without weakening host defence to infection.
Robert Moots, Professor of Rheumatology at the University of Liverpool and Director for Research and Development at Aintree University Hospital, said:
“We have shown that APPA has the potential to dampen down that bad inflammation that causes rheumatic diseases – but not impact on the crucial antimicrobial function of neutrophils. We have been waiting for too many years for such a selective drug.”
“Our results suggest a prime role for APPA in helping safely modify aggressive immune response, not only in the arthritis that I treat every day, but even, potentially, in COVID-19.”
Professor Steve Edwards, a neutrophil scientist on the project at University of Liverpool said: “Therapeutically targeting the harmful effects of neutrophils in inflammation, without interfering with their ability to fight off infections, has been a long-term goal of many scientists worldwide. At last, we may be able now to realise this goal.”
David Miles, CEO of AKLRD said: “These exciting results underpin the favourable clinical results observed in patients with Osteoarthritis, whilst also suggesting APPA has an important role to play in treating a broad range of conditions where inflammation is involved.”
Inflammation is an important process to defend against pathogens and injuries. A controlled acute inflammatory response is beneficial for the body. However, inflammation can become detrimental when the process is dysregulated.
Uncontrolled inflammation is underlying most chronic diseases such as cardiovascular disease, arthritis, asthma and type 2 diabetes mellitus [1] and is often linked to cancer development [1,2].
Inflammation is a complex process involving many mediators, with TNF-α, IL-6, IL-8, ROS and platelet activation being key players (Fig 1).
Even though Fig 1 addresses only a limited number of pathways, the complex nature of inflammation and the many mediators involved is apparent.
Interconnection of inflammatory pathways.
Five important processes in inflammation are combined into one interconnected pathway network. (TNF-α, green) TNF-α is part of the very extensive NF-κβ pathway [3]. TNF-α starts multiple signaling cascades by recruiting the tumor necrosis factor receptor 1 (TNFR1), which is subsequently recruiting the TNFR1 associated death domain (TRADD) [4]. TRADD on one side activates the caspase cascade which leads to apoptosis and ROS production [5]. On the other side, the core component complex IKKα/β (Iκα/β kinase) of the NF-κβ pathway is activated. The β part subsequently phosphorylates Iκβ which in turn activates NF-κβ [4]. This leads to translocation of NF-κβ dimers to the nucleus and upregulation of (among others) IL-6, IL-8, TNF-α, and manganese superoxide dismutase (Mn-SOD) [4–6]. (IL-6, pink) IL-6 is an important activator of the Janus kinase signal transducer and an activator of transcription [7]. The JAK/Stat pathway is involved in the upregulation of pro-inflammatory cytokines in inflammation, cell proliferation and tumorigenesis [6,8]. IL-6 binds to the IL-6 receptor (IL-6R), which in turn associates with the gp130 protein complex on the cell membrane and phosphorylates JAK. Only a few cell types express the IL-6R on the cell membrane, however, all cells have a soluble form of this receptor (sIL-6R) and the gp130 dimer, meaning that JAK/Stat signaling can be activated in essentially all cell types. The complexation of IL-6 with gp130 and consequently the phosphorylation of Stat3 is needed for a controlled inflammatory response [9,10]. Activated Stat3 dimerizes and translocates to the nucleus, where (among others) c-Myc and c-reactive protein (CRP) are upregulated [10]. Furthermore, activated Stat3 stimulates NF-κβ [2] and the Ras oncogene which is important in both the development of cancer [6,10] and stimulation of inflammation [6]. (IL-8, yellow) IL-8 is a pro-inflammatory chemokine whose expression is primarily regulated by NF-κβ. IL-8 binds to G-coupled protein receptor CXCR1/2, which in turn stimulates the Ras oncogene and promotes the nuclear translocation of Stat3 [11]. It is the most powerful human neutrophil chemoattractant and stimulates tumor growth. Furthermore, TNF-α and ROS are potent inducers of IL-8 production [12]. (ROS, blue) At an inflammatory site, ROS (which include superoxide radicals, nitric oxide and hydrogen peroxide [5]) are produced continuously (the oxidative burst) as one of the first lines of attack against pathogens [13]. ROS production is vital in acute inflammation, however, a too high production of ROS can cause DNA repair failure [6] and modifications in proteins [13], and are carcinogenic [5]. Intracellularly, most ROS are produced by the mitochondrial electron transport chain (ETC), which is also stimulated in response to TNF-α [5]. These ROS are important for apoptosis as well as cell maintenance, but also stimulate NF-κβ, inflammation and cancer [13]. ROS can also activate platelets [14]. (Platelets, orange) Platelets are derived from megakaryocytes, do not have a nucleus and are essential for hemostasis and thrombosis. However, platelets are also loaded with immune modulators, and can drive the inflammatory response. Platelets express NADPH oxidase (NOX) and are an important source of ROS [15]. Upon activation by thrombin or ROS, α-granules are secreted which contain (among others) fibrinogen, P-selectin and EGF (15) . f = fibrinogen, Psel = P-selectin, EGF = endothelial growth factor, α = α-granules, β3 = β-integrin receptors, LPS = lipopolysaccharide, Plt = platelet, Nu = nucleus.
To modulate inflammatory responses, a variety of anti-inflammatory drugs are used, which can be broadly categorized as non-steroidal anti-inflammatory drugs and steroids. Glucocorticoids (GCs) are the most robust anti-inflammatory agents known [16], widely used [17] and the most effective drugs in many chronic inflammatory and immune diseases [18,19].
In general, GCs decrease the transcription of pro-inflammatory cytokines and chemokines and increase the transcription of anti-inflammatory cytokines [17,18]. In addition, there are also non-genomic actions described for GCs [16].
Despite their strong anti-inflammatory activities, a variety of systemic side effects [17] can outweigh the benefits of GC treatment [20]. About 90% of patients with chronic GC treatment develop side effects, ranging from mild (acne) to severe (Cushing Syndrome) and even life threatening events (heart disease) [19].
Besides the adverse effects, individual patients can respond differently to GCs and, with chronic use, many patients develop a form of GC resistance [16]. A small number of patients is even completely resistant to initial GC therapy [18]. The need for alternative treatments is therefore substantial [17,18].
Particularly compounds with a different anti-inflammatory mechanism of action would be attractive. Such compounds might be found in the imminent field of natural products (NPs) because of their described anti-inflammatory bioactivity and great structural variety [21].
NPs show an enormous structural diversity [21,22]. They are important metabolites in plants and play key roles in antibiotic mechanisms, defense against herbivores, protection against UV radiation, nutrition and growth of the plant [23]. NPs are an important source for new drug development [21]. Indeed, half of the drugs in clinical use today is of natural origin [22,24].
In this study, in order to find active but less toxic alternatives for GCs, we have selected eight NPs because of their alleged anti-inflammatory properties and popularity. We have compared their therapeutic in vitro efficacy with that of the GC prednisolone (PLP, Fig 2.1).
We chose PLP, because it is an often prescribed GC which is with its intermediate potency a good representative of the GCs. As such, it serves as a good benchmark for the potency of the natural products in comparison to a strong anti-inflammatory compound. Although these eight NPs are well known, and their anti-inflammatory activity has been described, the head-to-head comparison in multiple anti-inflammatory assays, usually applied in pharmacology, is novel in phytomedicine.
Epigallocatechin gallate (EGCG, Fig 2.2), the main constituent in green tea leaves (Camellia sinensis) [25] has gained a lot of interest in the past decade [26]. It is a good radical scavenger with chemopreventive actions [23] and anti-inflammatory effects [26].
Berberine chloride (BBCl, Fig 2.3) is an alkaloid found in barberry plants (Berberis species) [27], which have a long history in traditional medicine [27,28]. BBCl is used in diabetes mellitus against insulin resistance [29]. Pharmacological properties of BBCl include antimicrobial, antidiarrheal [28], anti-inflammatory and anti-oxidant [21,28,29].
Curcumin (Cur, Fig 2.4) is a pigment [23] isolated from Curcuma longa which has been used in many ailments in traditional Indian medicine [30]. Cur has chemopreventive actions [23] and, despite its poor bioavailability, is used in the development of new anti-inflammatory and anti-cancer drugs [21].
Apocynin (Apo, Fig 2.5), isolated from Picrorhiza kurroa [31], is well known in traditional medicine [32], and inhibits NADPH oxidase [14,32] and platelet recruitment [14]. Apo dimerizes upon entering the cell, which enhances the effect [32]. Apocynin ester (Apo-e, Fig 2.6) is an ester form of apocynin making the molecule more lipophilic to increase oral bioavailability.
Paeonol (PN, Fig 2.7), the main phenolic compound in paeony roots (Paeonia species), is used in traditional medicine to treat inflammation [33,34]. Other reported properties of PN are anti-oxidant and apoptosis inducing effects [33].
Pterostilbene (PTS, Fig 2.8) is an anti-oxidant with anti-cancer properties found in berries [35,36]. PTS is an analogue of resveratrol, however with an enhanced oral bioavailability [35].
Pravastatin sodium (PSS, Fig 2.9) is the only included NP of microbial origin. PSS is part of the HMG-CoA reductase inhibitors, generally referred to as statins, which lower low density lipoprotein (LDL) levels [37] and have anti-inflammatory effects in patients with cardiovascular diseases [38].
One of the difficulties in interpreting the relative potency of NPs is the fact that most studies investigate a single molecular species in a single assay. In this benchmarking study, eight NPs are tested simultaneously in four in vitro anti-inflammatory assays representing five major pathways of inflammation (Fig 1) and are compared to the reference GC PLP.
Inhibition of secretion of pro-inflammatory cytokines TNF-α and IL-6 by macrophages, inhibition of secretion of pro-inflammatory chemokine IL-8 by colon epithelial cells, inhibition of ROS production by polymorphonuclear leukocytes and inhibition of platelet activation. From the collected results, anti-inflammatory profiles were established and compared to PLP to identify potent alternatives.
More information: A. L. Cross et al. APPA (apocynin and paeonol) modulates pathological aspects of human neutrophil function, without supressing antimicrobial ability, and inhibits TNFα expression and signalling, Inflammopharmacology (2020). DOI: 10.1007/s10787-020-00715-5
References
1. Medzhitov R. Origin and physiological roles of inflammation. Nature [Internet]. 2008. July 24 [cited 2014 Apr 15];454(7203):428–35. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18650913 [Abstract] [Google Scholar]
2. Atsumi T, Singh R, Sabharwal L, Bando H, Meng J, Arima Y, et al. Inflammation amplifier, a new paradigm in cancer biology. Cancer Res [Internet]. 2014. January 1 [cited 2014 May 27];74(1):8–14. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24362915 [Abstract] [Google Scholar]
16. Yang N, Ray DW, Matthews LC. Current concepts in glucocorticoid resistance. Steroids [Internet]. Elsevier Inc.; 2012. September [cited 2014 Jul 9];77(11):1041–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22728894 [Abstract] [Google Scholar]
17. Barnes PJ. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci (Lond) [Internet]. 1998. June [cited 2014 Jul 9];94(6):557–72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9854452 [Abstract] [Google Scholar]
18. Barnes PJ. Glucocorticosteroids: current and future directions. Br J Pharmacol [Internet]. 2011. May [cited 2014 Jul 9];163(1):29–43. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3085866&tool=pmcentrez&rendertype=abstract [Europe PMC free article] [Abstract] [Google Scholar]
19. Ericson-Neilsen W, Kaye A. Steroids: Pharmacology, Complications, and Practice Delivery Issues. Ochsner J [Internet]. 2014. January [cited 2014 Jul 9];14(2):203–7. Available from: http://www.ochsnerjournal.org/doi/abs/10.1043/1524-5012-14.2.203 [Europe PMC free article] [Abstract] [Google Scholar]
20. Fardet L, Flahault A, Kettaneh A, Tiev KP, Généreau T, Tolédano C, et al. Corticosteroid-induced clinical adverse events: Frequency, risk factors and patient’s opinion. Br J Dermatol [Internet]. 2007. July [cited 2014 Jul 9];157(1):142–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17501951 [Abstract] [Google Scholar]
21. Gautam R, Jachak SM. Recent developments in anti-inflammatory natural products. Med Res Rev [Internet]. 2009. September [cited 2014 Jul 14];29(5):767–820. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19378317 [Google Scholar]
22. Chin Y-W, Balunas MJ, Chai HB, Kinghorn a D. Drug discovery from natural sources. AAPS J [Internet]. 2006. January [cited 2014 Jul 14];8(2):E239–53. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3231566&tool=pmcentrez&rendertype=abstract [Europe PMC free article] [Abstract] [Google Scholar]
23. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed Engl [Internet]. 2011. January 17 [cited 2014 Mar 27];50(3):586–621. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21226137 [Abstract] [Google Scholar]
24. Paterson I, Anderson E a. Chemistry. The renaissance of natural products as drug candidates. Science [Internet]. 2005. October 21 [cited 2014 Jul 14];310(5747):451–3. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16239465 [Abstract] [Google Scholar]
25. Kada T, Kaneko K, Matsuzaki S, Matsuzaki T, Hara Y. Detection and chemical identification of natural bio-antimutagens. A case of the green tea factor. Mutat Res. 1985;150(1–2):127–32. [Abstract] [Google Scholar]
26. Riegsecker S, Wiczynski D, Kaplan MJ, Ahmed S. Potential benefits of green tea polyphenol EGCG in the prevention and treatment of vascular inflammation in rheumatoid arthritis. Life Sci [Internet]. Elsevier Inc.; 2013. September 3 [cited 2014 Jul 17];93(8):307–12. Available from: 10.1016/j.lfs.2013.07.006 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
27. Ikram M. A review on the chemical and pharmacological aspects of genus Berberis. Planta Med. 1975;28(4):353–8. [Abstract] [Google Scholar]
28. Pereira GC, Branco AF, Matos JAC, Pereira SL, Parke D, Perkins EL, et al. Mitochondrially targeted effects of berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse melanoma cells: comparison with direct effects on isolated mitochondrial fractions. J Pharmacol Exp Ther [Internet]. 2007. November [cited 2014 Apr 14];323(2):636–49. Available from: http://jpet.aspetjournals.org/content/early/2007/08/17/jpet.107.128017.short [Abstract] [Google Scholar]
29. Li Z, Geng Y-N, Jiang J-D, Kong W-J. Antioxidant and Anti-Inflammatory Activities of Berberine in the Treatment of Diabetes Mellitus. Evid Based Complement Alternat Med [Internet]. 2014. January [cited 2014 May 31];2014:289264 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3942282&tool=pmcentrez&rendertype=abstract [Europe PMC free article] [Abstract] [Google Scholar]
30. Kuttan R, Bhanumathy P, Nirmala K, George MC. Potential anticancer activity of turmeric (Curcuma longa). Cancer Lett [Internet]. 1985;29(2):197–202. Available from: http://www.sciencedirect.com/science/article/pii/0304383585901594 [Abstract] [Google Scholar]
31. Van den Worm E, Beukelman CJ, Van den Berg AJ, Kroes BH, Labadie RP, Van Dijk H. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils. Eur J Pharmacol [Internet]. 2001. December 21 [cited 2014 Apr 25];433(2–3):225–30. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0014299901015163 [Abstract] [Google Scholar]
32. Stefanska J, Pawliczak R. Apocynin: molecular aptitudes. Mediators Inflamm [Internet]. 2008. January [cited 2014 Mar 23];2008:106507 Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2593395&tool=pmcentrez&rendertype=abstract [Europe PMC free article] [Abstract] [Google Scholar]
33. Huang H, Chang EJ, Lee Y, Kim JS, Kang SS, Kim HH. A genome-wide microarray analysis reveals anti-inflammatory target genes of paeonol in macrophages. Inflamm Res [Internet]. 2008. April [cited 2014 Jul 23];57(4):189–98. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18363035 [Abstract] [Google Scholar]
34. Chen N, Liu D, Soromou LW, Sun J, Zhong W, Guo W, et al. Paeonol suppresses lipopolysaccharide-induced inflammatory cytokines in macrophage cells and protects mice from lethal endotoxin shock. Fundam Clin Pharmacol [Internet]. 2014. June [cited 2014 Jul 23];28(3):268–76. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23413967 [Abstract] [Google Scholar]
35. McCormack D, McFadden D. Pterostilbene and cancer: current review. J Surg Res [Internet]. Elsevier Inc; 2012. April [cited 2013 Oct 4];173(2):e53–61. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22099605 [Abstract] [Google Scholar]
36. Hsu C-LL, Lin Y-JJ, Ho C-TT, Yen G-CC. The inhibitory effect of pterostilbene on inflammatory responses during the interaction of 3T3-L1 adipocytes and RAW 264.7 macrophages. J Agric Food Chem [Internet]. 2013. January 23 [cited 2014 Jul 23];61(3):602–10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23268743 [Abstract] [Google Scholar]
37. Tobert J a. Lovastatin and beyond: the history of the HMG-CoA reductase inhibitors. Nat Rev Drug Discov [Internet]. 2003. July [cited 2014 Jul 10];2(7):517–26. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12815379 [Abstract] [Google Scholar]
38. Krane V, Wanner C. Statins, inflammation and kidney disease. Nat Rev Nephrol [Internet]. Nature Publishing Group; 2011. July [cited 2014 Jul 9];7(7):385–97. Available from: 10.1038/nrneph.2011.62 [Abstract] [CrossRef] [Google Scholar]
