The Concise Guide to PHARMACOLOGY 2023/24: Enzymes.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Intracellular immunoglobulin A (icIgA) in protective immunity and vaccines.

Virus neutralization at respiratory mucosal surfaces is important in the prevention of infection. Mucosal immunity is mediated mainly by extracellular secretory immunoglobulin A (sIgA) and its role has been well studied. However, the protective role of intracellular specific IgA (icIgA) is less well defined. Initially, in vitro studies using epithelial cell lines with surface expressed polymeric immunoglobulin receptor (pIgR) in transwell culture chambers have shown that icIgA can neutralize influenza, parainfluenza, HIV, rotavirus and measles viruses. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. Co-localization of specific icIgA with influenza virus in patients' (virus culture positive) respiratory epithelial cells using well-characterized antisera was initially reported in 2018. This review provides a summary of in vitro studies with icIgA on colocalization and neutralization of the above five viruses. Two other highly significant respiratory infectious agents with severe global impacts viz. SARS-2 virus (CoViD pandemic) and the intracellular bacterium-Mycobacterium tuberculosis-are discussed. Further studies will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal infections. This will inform the design of more effective vaccines against infectious agents transmitted via the mucosal route.

TRPM8 indicates poor prognosis in colorectal cancer patients and its pharmacological targeting reduces tumour growth in mice by inhibiting Wnt/ß-catenin signalling.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin type-8 (TRPM8) is a cold-sensitive cation channel protein belonging to the TRP superfamily of ion channels. Here, we reveal the molecular mechanism of TRPM8 and its clinical relevance in colorectal cancer (CRC). EXPERIMENTAL APPROACH: TRPM8 expression and its correlation with the survival rate of CRC patients was analysed. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted in CRC patients. TRPM8 functional role was assessed by using Trpm8-/- mice in models of sporadic and colitis-associated colon cancer. TRPM8 pharmacological targeting by WS12 was evaluated in murine models of CRC. KEY RESULTS: TRPM8 is overexpressed in colon primary tumours and in CD326+ tumour cell fraction. TRPM8 high expression was related to lower survival rate of CRC patients, Wnt-Frizzled signalling hyperactivation and adenomatous polyposis coli down-regulation. In sporadic and colitis-associated models of colon cancer, either absence or pharmacological desensitization of TRPM8 reduced tumour development via inhibition of the oncogenic Wnt/ß-catenin signalling. TRPM8 pharmacological blockade reduced tumour growth in CRC xenograft mice by reducing the transcription of Wnt signalling regulators and the activation of ß-catenin and its target oncogenes such as C-Myc and Cyclin D1. CONCLUSION AND IMPLICATIONS: Human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival. Animal experiments demonstrate TRPM8 involvement in colon cancer pathophysiology and its potential as a drug target for CRC.

Planning experiments: Updated guidance on experimental design and analysis and their reporting III.

Scientists who plan to publish in British Journal of Pharmacology (BJP) must read this article before undertaking a study. This editorial provides guidance for the design of experiments. We have published previously two guidance documents on experimental design and analysis (Curtis et al., 2015; Curtis et al., 2018). This update clarifies and simplifies the requirements on design and analysis for BJP manuscripts. This editorial also details updated requirements following an audit and discussion on best practice by the BJP editorial board. Explanations for the requirements are provided in the previous articles. Here, we address new issues that have arisen in the course of handling manuscripts and emphasise three aspects of design that continue to present the greatest challenge to authors: randomisation, blinded analysis and balance of group sizes.

Future directions for the discovery of natural product-derived immunomodulating drugs: an IUPHAR positional review.

Drug discovery from natural sources is going through a renaissance, having spent many decades in the shadow of synthetic molecule drug discovery, despite the fact that natural product-derived compounds occupy a much greater chemical space than those created through synthetic chemistry methods. With this new era comes new possibilities, not least the novel targets that have emerged in recent times and the development of state-of-the-art technologies that can be applied to drug discovery from natural sources. Although progress has been made with some immunomodulating drugs, there remains a pressing need for new agents that can be used to treat the wide variety of conditions that arise from disruption, or over-activation, of the immune system; natural products may therefore be key in filling this gap. Recognising that, at present, there is no authoritative article that details the current state-of-the-art of the immunomodulatory activity of natural products, this in-depth review has arisen from a joint effort between the International Union of Basic and Clinical Pharmacology (IUPHAR) Natural Products and Immunopharmacology Sections, with contributions from a number of world-leading researchers in the field of natural product drug discovery, to provide a "position statement" on what natural products has to offer in the search for new immunomodulatory argents. To this end, we provide a historical look at previous discoveries of naturally occurring immunomodulators, present a picture of the current status of the field and provide insight into the future opportunities and challenges for the discovery of new drugs to treat immune-related diseases.

N-Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth.

BACKGROUND AND PURPOSE: N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N-acylethanolamines (NAEs) and its pharmacological inhibition has beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism, whereas its contribution to colorectal cancer (CRC) is unknown to date. EXPERIMENTAL APPROACH: CRC xenograft and azoxymethane models were used to assess the in vivo effect of NAAA inhibition. Further, the tumour secretome was evaluated by an oncogenic array, CRC cell lines were used for in vitro studies, cell cycle was analysed by cytofluorimetry, NAAA was knocked down with siRNA, human biopsies were obtained from surgically resected CRC patients, gene expression was measured by RT-PCR and NAEs were measured by LC-MS. KEY RESULTS: The NAAA inhibitor AM9053 reduced CRC xenograft tumour growth and counteracted tumour development in the azoxymethane model. NAAA inhibition affected the composition of the tumour secretome inhibiting the expression of EGF family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR-α and TRPV1. AM9053 induced cell cycle arrest in the S phase associated with cyclin A2/CDK2 down-regulation. NAAA knock-down mirrored the effects of NAAA inhibition with AM9053. NAAA expression was down-regulated in human CRC tissues, with a consequential augmentation of NAE levels and dysregulation of some of their targets. CONCLUSION AND IMPLICATIONS: Our results show novel data on the functional importance of NAAA in CRC progression and the mechanism involved. We propose that this enzyme is a valid drug target for the treatment of CRC growth and development.

Guinea Pig Model of Mycobacterium tuberculosis Infection.

Guinea pigs have been used as a model for Mycobacterium tuberculosis infection for many years and have been more recently adopted as a model for testing new tuberculosis (TB) vaccines. From the time of Robert Koch, who used guinea pigs to test theories about the newly discovered pathogen, the guinea pig has modeled active human infections, as it is susceptible to infection with low numbers of organisms. This article describes the modern use of the guinea pig to examine the pathology of the disease and the protocols used to examine specific outcomes associated with aerosol infection with virulent M. tuberculosis. The guinea pig is used extensively to investigate the ability of new TB vaccines to reduce TB disease, and two models have been employed. The first is the long-term disease model, in which vaccinated guinea pigs are monitored for disease after infection, and the second is the short-term assessment of mycobacterial burden model, which can determine the ability of a vaccine to reduce organism burden. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of virulent Mycobacterium tuberculosis seed stocks for animal infections Basic Protocol 2: Preparation of virulent Mycobacterium tuberculosis working stocks for animal infections Basic Protocol 3: Preparation of M. tuberculosis for aerosol infection of guinea pigs Basic Protocol 4: Injection of guinea pigs Basic Protocol 5: Blood collection from live guinea pigs Basic Protocol 6: Guinea pig euthanasia.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Enzymes.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Palmitoylethanolamide Reduces Colon Cancer Cell Proliferation and Migration, Influences Tumor Cell Cycle and Exerts In Vivo Chemopreventive Effects.

Palmitoylethanolamide (PEA) is an endogenous fatty acid amide related to the endocannabinoid anandamide. PEA exerts intestinal anti-inflammatory effects, but knowledge of its role in colon carcinogenesis is still largely fragmentary. We deepened this aspect by studying the effects of PEA (ultramicronized PEA, um-PEA) on colon cancer cell proliferation, migration and cell cycle as well as its effects in a murine model of colon cancer. Results showed that um-PEA inhibited tumor cell proliferation via peroxisome proliferator-activated receptor α and G protein-coupled receptor 55, induced cell cycle arrest in the G2/M phase, possibly through cyclin B1/CDK1 upregulation, and induced DNA fragmentation. Furthermore, um-PEA reduced tumor cell migration by reducing MMP2 and TIMP1 expression. In vivo administration of um-PEA exerted beneficial effects in the azoxymethane model of colonic tumors, by reducing the number of preneoplastic lesions and tumors. Collectively, our findings provide novel proofs on the effects of um-PEA in colon carcinogenesis.

A Glucuronic Acid-Palmitoylethanolamide Conjugate (GLUPEA) Is an Innovative Drug Delivery System and a Potential Bioregulator.

Palmitoylethanolamide (PEA) is an endogenous anti-inflammatory lipid mediator and a widely used nutraceutical. In this study, we designed, realized, and tested a drug-carrier conjugate between PEA (the active drug) and glucuronic acid (the carrier). The conjugate, named GLUPEA, was characterized for its capability of increasing PEA levels and exerting anti-inflammatory activity both in vitro and in vivo. GLUPEA treatment, compared to the same concentration of PEA, resulted in higher cellular amounts of PEA and the endocannabinoid 2-arachidonoyl glycerol (2-AG), and increased 2-AG-induced transient receptor potential vanilloid type 1 (TRPV1) channel desensitization to capsaicin. GLUPEA inhibited pro-inflammatory monocyte chemoattractant protein 2 (MCP-2) release from stimulated keratinocytes, and it was almost as efficacious as ultra-micronized PEA at reducing colitis in dinitrobenzene sulfonic acid (DNBS)-injected mice when using the same dose. GLUPEA is a novel pro-drug able to efficiently mimic the anti-inflammatory and endocannabinoid enhancing actions of PEA.

Efficacy of combined therapy with fish oil and phytocannabinoids in murine intestinal inflammation.

Fish oil (FO) and phytocannabinoids have received considerable attention for their intestinal anti-inflammatory effects. We investigated whether the combination of FO with cannabigerol (CBG) and cannabidiol (CBD) or a combination of all three treatments results in a more pronounced intestinal antiinflammatory action compared to the effects achieved separately. Colitis was induced in mice by 2,4-dinitrobenzenesulfonic acid (DNBS). CBD and CBG levels were detected and quantified by liquid chromatography coupled with time of flight mass spectrometry and ion trap mass spectrometry (LC-MS-IT-TOF). Endocannabinoids and related mediators were assessed by LC-MS. DNBS increased colon weight/colon length ratio, myeloperoxidase activity, interleukin-1ß, and intestinal permeability. CBG, but not CBD, given by oral gavage, ameliorated DNBS-induced colonic inflammation. FO pretreatment (at the inactive dose) increased the antiinflammatory action of CBG and rendered oral CBD effective while reducing endocannabinoid levels. Furthermore, the combination of FO, CBD, and a per se inactive dose of CBG resulted in intestinal anti-inflammatory effects. Finally, FO did not alter phytocannabinoid levels in the serum and in the colon. By highlighting the apparent additivity between phytocannabinoids and FO, our preclinical data support a novel strategy of combining these substances for the potential development of a treatment of inflammatory bowel disease.

Botanical drugs and supplements affecting the immune response in the time of COVID-19: Implications for research and clinical practice.

In times of health crisis, including the current COVID-19 pandemic, the potential benefit of botanical drugs and supplements emerges as a focus of attention, although controversial efficacy claims are rightly a concern. Phytotherapy has an established role in everyday self-care and health care, but, since botanical preparations contain many chemical constituents rather than single compounds, challenges arise in demonstrating efficacy and safety. However, there is ample traditional, empirical, and clinical evidence that botanicals can offer some protection and alleviation of disease symptoms as well as promoting general well-being. Newly emerging viral infections, specifically COVID-19, represent a unique challenge in their novelty and absence of established antiviral treatment or immunization. We discuss here the roles and limitations of phytotherapy in helping to prevent and address viral infections, especially regarding their effects on immune response. Botanicals with a documented immunomodulatory, immunostimulatory, and antiinflammatory effects include adaptogens, Boswellia spp., Curcuma longa, Echinacea spp., Glycyrrhiza spp., medicinal fungi, Pelargonium sidoides, salicylate-yielding herbs, and Sambucus spp. We further provide a clinical perspective on applications and safety of these herbs in prevention, onset, progression, and convalescence from respiratory viral infections.

Fish Oil, Cannabidiol and the Gut Microbiota: An Investigation in a Murine Model of Colitis.

Inflammatory bowel disorders can be associated with alterations in gut microbiota (dysbiosis) and behavioral disturbances. In experimental colitis, administration of fish oil (FO) or cannabinoids, such as cannabidiol (CBD), reduce inflammation. We investigated the effect of combined FO/CBD administration on inflammation and dysbiosis in the dextran sulphate sodium (DSS) model of mouse colitis, which also causes behavioral disturbances. Colitis was induced in CD1 mice by 4% w/v DSS in drinking water for five consecutive days followed by normal drinking water. FO (20-75 mg/mouse) was administered once a day starting two days after DSS, whereas CBD (0.3-30 mg/kg), alone or after FO administration, was administered once a day starting 3 days after DSS, until day 8 (d8) or day 14 (d14). Inflammation was assessed at d8 and d14 (resolution phase; RP) by measuring the Disease Activity Index (DAI) score, change in body weight, colon weight/length ratio, myeloperoxidase activity and colonic interleukin (IL)-1ß (IL-1ß), IL-10, and IL-6 concentrations. Intestinal permeability was measured with the fluorescein isothiocyanate-dextran. Behavioral tests (novel object recognition (NOR) and light/dark box test) were performed at d8. Fecal microbiota composition was determined by ribosomal 16S DNA sequencing of faecal pellets at d8 and d14. DSS-induced inflammation was stronger at d8 and accompanied by anxiety-like behavior and impaired recognition memory. FO (35, 50, 75 mg/mouse) alone reduced inflammation at d8, whereas CBD alone produced no effect at any of the doses tested; however, when CBD (3, 10 mg/kg) was co-administered with FO (75 mg/mouse) inflammation was attenuated. FO (20 mg/mouse) and CBD (1 mg/kg) were ineffective when given alone, but when co-administered reduced all inflammatory markers and the increased intestinal permeability at both d8 and d14, but not the behavioral impairments. FO, CBD, and their combination affected gut bacteria taxa that were not affected by DSS per se. Akkermansia muciniphila, a species suggested to afford anti-inflammatory action in colitis, was increased by DSS only at d14, but its levels were significantly elevated by all treatments at d8. FO and CBD co-administered at per se ineffective doses reduce colon inflammation, in a manner potentially strengthened by their independent elevation of Akkermansia muciniphila.

Immunization with Mycobacterium tuberculosis-Specific Antigens Bypasses T Cell Differentiation from Prior Bacillus Calmette-Guérin Vaccination and Improves Protection in Mice.

Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.

Pulmonary mucosal immunity mediated through CpG provides adequate protection against pulmonary Mycobacterium tuberculosis infection in the mouse model. A role for type I interferon.

Toll-Like Receptor (TLR) 9 stimulation is required for induction of potent immune responses against pathogen invasion. The use of unmethylated CpG as adjuvants in vaccines provides an excellent means of stimulating adaptive immunity. Our data demonstrate that CpG-C provided prolonged immune responses in the mouse model of tuberculosis when formulated with liposomes and the Mycobacterium tuberculosis antigen ESAT-6. A reduction in the mycobacterial burden was best achieved when administered as an intranasal vaccine and was dependent on type I interferon (IFN). There was a significant difference between CpG-C inoculated wild type and IFN-αR1-/- mice, indicating that type I IFN plays a role in the immune response following CpG-C inoculation. Further analysis showed that early NK cell presence was not an absolute requirement, although elevated IFN-γ levels were detected in the lungs of mice within 48 h. The reduction in mycobacterial burden was MyD88-independent as CpG-C inoculated MyD88-/- mice showed comparable mycobacterial burdens to wild type mice with no detriment due to the lack of MyD88. Together our data show that pulmonary stimulation of TLR9 bearing antigen presenting cells resulted in the induction of protective immunity against M. tuberculosis infection that was dependent on type I IFN signaling.

Characterizing the BCG Induced Macrophage and Neutrophil Mechanisms for Defense Against Mycobacterium tuberculosis.

The live attenuated Mycobacterium bovis strain, Bacille Calmette Guérin (BCG) is a potent innate immune stimulator. In the C57BL/6 mouse model of tuberculosis, BCG vaccination leads to a significant reduction of Mycobacterium tuberculosis burden after aerogenic infection. Our studies indicated that BCG induced protection against pulmonary tuberculosis was independent of T cells and present as early as 7 days after vaccination. This protection showed longevity, as it did not wane when conventional T cell and TNF-α deficient mice were infected 30 days post-vaccination. As BCG induced mycobacterial killing after 7 days, this study investigated the contributions of the innate immune system after BCG vaccination to better understand mechanisms required for mycobacterial killing. Subcutaneous BCG inoculation resulted in significant CD11b+F4/80+ monocyte subset recruitment into the lungs within 7 days. Further studies revealed that killing of mycobacteria was dependent on the viability of BCG, because irradiated BCG did not have the same effect. Although others have identified BCG as a facilitator of trained innate immunity, we found that BCG reduced the mycobacterial burden in the absence of mechanisms required for trained innate immunity, highlighting a role for macrophages and neutrophils for vaccine induced killing of M. tuberculosis.