Respiratory diseases Flashcards
Asthma and COPD
Extrinsic asthma is allergic-type hypersensitivity to allergens.
Intrinsic asthma is non-allergic sensitivity to irritants.
Asthma involves airway inflammation and bronchoconstriction, which can be reversed by beta agonists.
Airway remodelling leads to hyperresponsiveness due to histamine release.
Preventative treatment includes steroids and leukotriene receptor antagonists (LTRAs).
COPD is mostly caused by cigarette smoke or Alpha-1-antitrypsin deficiency.
Comprises emphysema and bronchitis.
Involves airway inflammation and obstruction that is not readily reversible.
Some patients have difficult to treat (DTT), uncontrolled or severe asthma.
- DTT - asthma is uncontrolled despite prescribing medium or high-dose controller treatment
- Uncontrolled - frequent symptoms and exacerbations
- Severe - asthma that is uncontrolled despite maximal optimised theraphy (high-dose ICS-LABA) and treatment of contributory factors or worsens when high-dose treatment is decreased.
COPD is DTT and steroid resistance can develop.
Novel treatment is required for DTT asthma and COPD.
Asthma pathology
Asthma pathology is complex.
Mast cells and IgE release play a role.
There is an imbalance in T cell types, particularly in the Th1/Th2 population as Th2 numbers increase. There is also a deficiency in Tregs.
A range of different chemical medicators are involved.
Asthma is a heterogeneous disease, which means that patients may respond differently to different treatments. There’s a convergence of different pathophysiologic mechanisms that lead to asthma.
There are different phenotypes, which is the outward manifestation of the disease.
There are different endotypes, which are the molecular mechanisms that lead to such phenotypes.
The current concept on the heterogenous phenotypes of asthma is that several inflammatory subtypes are involved:
- Eosinophilic - may be due to allergic and non-allergic conditions
- Mixed eosinophilic and neutrophilic - elevated levels of both immune cells
- Neutrophilic
- Paucigranulocytic – absence of granulocytic inflammation
The different phenotypes fall into different ranges of the spectrum of endotypes.
- Type 2/T2 High – characterised by upregulation of T2 immune pathways (e.g. IL-4 and IL-13) and eosinophilic airway inflammation.
- Non-Type 2/T2-low – lacks signature biomarkers. It exists in the absence of T2-high or eosinophilic inflammation. It includes neutrophilic and paucigranulocytic subtypes.
Biologics for asthma
Omalizumab - anti-IgE monocloncal antibody approved in 2003
Anti-IL-5 antibodies include:
- Mepolizumab (2015)
- Reslizumab (2016)
- Benralizumab (2017)
Dupilumab is an anti-IL-4/13 antibody approved in 2017 for asthma and 2024 for COPD.
Tezepelumab is an anti-TSLP antibody approved in 2021.
Thymic stromal lymphopoietin (TSLP) is a cytokine produced by epithelial cells, keratinocytes, stromal cells, and allergen-activated basophils during exposure to inhaled environmental allergens and pollutants.
Benralizumab and IL-5
IL-5 is produced by Th2 lymphocytes in response to activation by antigen presenting cells (APCs), like macrophages or dendritic cells.
The IL-5 receptor is composed of an α subunit (IL-5Rα), which is specific for IL-5, and a common beta (βc) chain, which can bind IL-5 and IL-3.
IL-5 binding to IL-5Rα induces the dimerization of α and βc receptor components.
IL-5 stimulates eosinophil differentiation/maturation, survival, proliferation, adhesion, chemoattraction, and degranulation.
IL-5 is also involved in damage to the epithelial layer through release of cytotoxic proteins.
Inhibiting signalling therefore protects the airway epithelium.
Benralizumab is used in uncontrolled asthma, particularly eosinophilic asthma.
Benralizumab binds to interleukin-5 receptor alpha (IL-5Rα), which prevents IL-5 from activating eosinophils and basophils.
It binds IL-5Rα through its antigen binding site and to the FcγRIIIα receptor via the Fc (constant) region on natural killer cells.
It therefore has dual effects:
- Prevents IL-5 binding to the receptor, hence preventing activation of the receptor
- Attracts NK cells to eosinophils leading to apoptosis of cells.
Tezepelumab and TSLP
Tezepelumab binds to TSLP (thymic stromal lymphopoietin), an epithelial-derived cytokine.
TSLP is part of a class of cytokines known as alarmins produced by the airway epithelium in response to a perceived threat, such as inhaled allergens. Expression is increased in the airways of patients with asthma.
TSLP is a major mediator of eosinophilic inflammation.
It binds to the TSLP receptor, which forms heterodimer with the IL-7 receptor to stimulate inflammatory responses.
This stimulates the differentiation of naïve T cells to Th2 cells.
It also promotes airway remodelling, including smooth muscle proliferation.
Tezepelumab prevents TSLP from binding to its receptor, so no dimer is formed.
Asthma burden
Many patients with difficult-to-treat or severe asthma have inadequate response to biologic treatment and oral corticosteroids and fail to achieve asthma control.
Exacerbation rates in study populations with anti-IgE, anti-IL-5, anti-IL-5Rα, and anti-IL-4Rα treatment are reduced by approximately 50% only.
Existing biologics target immunological pathways that are downstream in the type 2 inflammatory cascade. This may explain why exacerbations are only partly reduced - it may be worth blocking more upstream targets that would result in the inhibition of multiple inflammatory pathways.
Type 2 airway inflammation results from several inflammatory signals in addition to IL-5. Consequences of inflammation driven by IL-13, such as increased fractional exhaled NO (FeNO), may remain unchanged with IL-5 treatment.
The broad inflammatory response involving IL-4, IL-5, and IL-13 that ultimately results in the classic features of exacerbations, including eosinophilic inflammation, mucus production and bronchospasm, is initiated by the release of ‘alarmins’, such as TSLP, IL-33, and IL-25, which are released from the airway epithelium in response to triggers.
Bitter taste receptor agonists
Bitter taste receptors, TAS2Rs, are present in the mouth, but also in the airways, where they play a role in detecting harmful substances, including potentially toxic or irritating substances.
In asthma, activation of TAS2R triggers a protective response which causes relaxation of airways smooth muscle and increased mucociliary clearance.
It is therefore a possible novel target, but agonists are not very potent in controlling asthma symptoms.
Rho kinase inhibitors
Rho kinases are activated by RhoA.
Activated Rho kinase inhibits myosin phosphatase, an enzyme that normally dephosphorylates MLC to DECREASE smooth muscle contraction.
As Rho kinase prevents the phosphorylation of myosin light chains, it promotes contraction at low levels of calcium (calcium sensitisation).
Rho kinase sustains smooth muscle contraction, which contributes to airway narrowing and hyperresponsiveness.
Rho kinase also plays a role in airway remodelling as it stimulates the proliferation of fibroblasts and enhances their ability to produce extracellular matrix components such as collagen. Excess collagen deposition contributes to airway remodelling, leading to increased airway stiffness and reduced lung function.
Rho kinase is also activated in eosinophils, so inhibitors reduce the recruitment of eosinophils.
Y27632 is an inhibitor that, when inhaled, produces rapid bronchodilation, which is maintained for 8 hours.
It has been used as a research tool and tested in animal species but not humans.
Calcilytics
Calcilytics are drugs that target the calcium-sensing receptor (CaSR).
CaSRs are GPCRs that mediate the intracellular responses to extracellular calcium levels.
They can be coupled to multiple G proteins:
- Gq - stimulates calcium
- Gi - inhibits cAMP - cAMP causes smooth muscle relaxation, so inhibition causes contraction.
- G12,13 - RhoA signalling pathway → contraction
CaSRs are activated by calcium and involved in regulating smooth muscle contraction, inflammation and mucus secretion.
Positive allosteric modulators (PAMs) enhance the sensitivity of the channels to calcium and are therefore called calcimimetics.
Negative allosteric modulators (NAMs) reduce sensitivity to calcium and are called calcilytics.
In asthma, there is an over-expression of CaSRs, which can lead to hyperresponsiveness.
NPS2143 is a calcilytic that reduces calcium levels in ASM cells from asthmatics but not from control patients. This suggests a selective effect in asthmatics.
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NPS2143 improves ovalbumin-induced airway hyperresponsiveness in mice.
It also reduces the production of OVA-specific IgE and IgG1 in serum and decreased the number of eosinophils and lymphocytes in bronchoalveolar lavage fluid (BALF), which was associated with reduced CaSR expression in lung tissues.
Additionally, NPS2143 alleviated the peribronchial and perivascular inflammatory cell infiltration, goblet cell hyperplasia, and airway mucus hypersecretion.
Drugs affecting leukotrienes and prostaglandins
Leukotrienes and prostaglandins are involved in the early phase of asthma, which causes bronchoconstriction.
Chemokines and chemotaxis are involved in attracting leukocytes, playing a role in the late phase of asthma.
Leukotrienes and prostaglandins are produced by enzymatic reactions. PLA2 releases arachidonic acid from the plasma membrane. This is then converted into leukotrienes by lipoxygenase and prostaglandins by cyclooxygenase.
Zileuton is a lipoxygenase inhibitor in clinical use in the US.
Setileuton is a similar compound which got up to phase II trials. It improves FEV1 and reduces the recruitment of immune cells, but it causes hepatotoxicity.
PGD2 effects are mediated through DP1 and DP2 receptors.
DP2 receptors are expressed on Th2 cells, mast cells, basophils and eosinophils.
Selective DP2 receptor antagonists include Timapiprant (OC000459), setipiprant and fevipiprant.
These inhibit activation of Th2 cells and eosinophils.
Setipiprant is in phase II clinical trials. It causes a reduction of hyperresponsiveness, but the observable benefit was no better than existing therapies, so it was not progressed.
Fevipiprant causes a reduction in eosinophil numbers in patients with asthma.
PDE inhibition
Phosphodiesterases (PDEs) are enzymes that catalyse the breakdown of cAMP and cGMP into their inactive forms.
In asthma treatment, we want to increase cAMP to allow smooth muscle relaxation.
The PDE4 isoform plays a crucial role in regulating smooth muscle contraction in the airways.
By degrading cAMP, PDE4 reduces intracellular cAMP levels, leading to smooth muscle contraction and bronchoconstriction.
PDE inhibitors play a significant role in asthma management by targeting specific isoforms of the PDE enzyme family.
PDE3 selective inhibitors like Enoximone induce airway smooth muscle relaxation and bronchodilation in asthmatic patients. However, inhibition of PDE3 in cardiac muscle increases the force of contraction, which limits its use.
PDE4 is expressed on mast cells and expression levels are raised in asthma and COPD.
PDE4 inhibitors, like Roflumilast, reduce the release of inflammatory mediators from mast cells.
PDE4 is also found in smooth muscle, but no clinical effect on bronchodilation has been shown with any inhibitors.
Roflumilast is therefore not approved in asthma, but it is approved for the treatment of COPD.
Apremilast and Roflumilast have been shown to reduce eosinophil recruitment through the reduction in cytokine production in Th2 cells.
Tanimilast (CHF6001) has shown reduction in pro-inflammatory mediators in patients with asthma in clinical trials. This supports the role of PDE4 in inflammatory responses.
Drugs that inhibit both PDE3 and PDE4 could reduce airway inflammation and cause bronchodilation.
Ensifentrine is a dual inhibitor that shows airway smooth muscle relaxation and a reduction in eosinophil numbers. It is approved for COPD.
It was found to improve lung function in clinical trials, so it may also gain approval for asthma.
Delivery through inhalers would reduce cardiac side effects.
Omalizumab (Xolair)
Omalizumab is a monoclonal antibody against free IgE used in severe, allergic asthma that cannot be controlled by steroids.
It prevents IgE from binding to mast cells thus preventing allergen-induced mediator release.
It is administered via subcutaneous injections every 2-4 weeks.
Cystic fibrosis
Cystic fibrosis is a heritable disorder affecting ion transporters in epithelial cells.
Respiratory, hepatobiliary, gastrointestinal and reproductive tracts and the pancreas are affected.
In cystic fibrosis, there are defects in the chloride transporter Cystic Fibrosis transmembrane conductance regulator (CFTR) leading to reduced Cl- and H2O transport.
This causes thicker mucus secretions that are hard to clear with obstruction and destruction of exocrine glandular ducts.
Recurrent infections occur as pathogens become trapped in the mucus but this cannot be cleared.
Disease characteristics include pancreatic exocrine insufficiency causing reduced or absent secretions. Impairment of pancreatic enzyme secretions causes people with CF to develop diabetes. Pancreatic enzyme replacement treatment (PERT) with enzymes like pancreatin is used. These are inactivated by gastric acid therefore given with food.
Patients living longer develop complications including diabetes (20%), liver disease, and osteoporosis.
Management of cystic fibrosis
The aim in cystic fibrosis management is to clear the viscous mucus from the airways, treat respiratory infection and improve respiratory function.
The lung disease is treated with:
- Physiotherapy like pressure massages to help push the mucus out.
- Antibiotics
- Corticosteroids
- Bronchodilators
- Dornase alfa administered using nebuliser. This is a synthetic version of the enzyme which cleaves extracellular DNA, digesting DNA released from dying neutrophils in the airway which otherwise contributes to mucus viscosity. (Neutrophils are produced to combat the infections, but these then die and release their DNA.)
Cystic Fibrosis transmembrane conductance regulator (CFTR) transporters
Reduced CFTR transporter function leads to symptoms of CF.
Drugs that increase channel activity should improve function.
There are different genetic variations observed in different patients.
A number of patients with CF have a “gating” variant in function of CFTR (F508del mutation).
CFTR localises to the cell membrane but is not activated by cAMP, so is not functional.
The channel is present but opening is impaired - it either does not open fully or it doesn’t open at all.
Phenotypic drug discovery
Target-based drug discovery (TDD) involves identifying a target involved in disease and screening compounds against that target.
In phenotypic drug discovery (PDD), compounds are screened to identify those that replicate a particular phenotype, e.g. healthy cell readout.
TDD requires only the biological target, while PDD requires a full biological system, such as human cells, tissue, animals or patients.
TDD is cost effective. It has the potential to generate lots of hits, but also lots of false positives.
A biological system is more complex than just the target, e.g. it considers feedback loops. Off-target effects can also complicate the overall effect.
Examples of drugs identified by phenotypic screening include Ivacaftor, an oral CFTR potentiator identified using gain-of-function assays in cell cultures.
PDD can reveal novel drug targets, for example targeting the folding of a protein.
Phenotypic screening has allowed us to develop cystic fibrosis drugs that affect the cause of the disease rather than just the symptoms.
CFTR modulators
Ivacaftor is a CFTR potentiator identified using PDD. It binds to CFTR and keeps the channel open for longer, enabling chloride transport.
Tezacaftor improves folding of the F508del-CFTR protein, so there is more functional protein at the membrane.
It is known as a CFTR corrector.
- CFTR is glycosylated as part of post-translational modification. Tezacaftor binds transmembrane domain 1 (TMD1), which increases the amount of glycosylated CFTR present at the membrane. In CF, mutations in the TMD1 binding site reduces the amount of glycosylated CFTR. The mechanism of action is not entirely clear, but TMD1 probably stabilises the folded protein.
Lumacaftor acts as a chaperone during protein folding, so it increases the number of CFTR proteins that are trafficked to the cell surface.
It also binds to the TMD1, but at a slightly different region. The binding site is near to where the protein interacts with the plasma membrane.
- The CFTR mutation causes defective folding of the protein. The misfolded protein remains in the endoplasmic reticulum and is degraded.
- TMD1 is folded after synthesis of the receptor (post-translationally). Lumacaftor is thought to stabilise TMD1 during folding so more protein gets transported to the membrane.
Combinations of drugs are more effective. For example, Lumacaftor and Ivacaftor mean that there is more protein at the cell surface, and these channels stay open for longer.
Elexacaftor is probably a CFTR corrector and CFTR potentiator. It corrects the misfolding AND keeps the ion channel open for longer.
Cough
Cough is a protective mechanism.
The cough reflex is initiated by sensory nerves in the upper airways, triggered by irritants.
Sensory nerves stimulate the diaphragm, abdominal muscles and intercostal muscles.
Chronic cough in respiratory diseases like asthma and COPD can be debilitating. For example, they can keep the patient awake at night. It is also very tiring as a lot of muscles are used.
Sensory nerves and cough
Sensory nerves are upregulated by inflammation.
They may be hypersensitive in asthmatics.
Sensory nerves have a potential role in asthma-induced cough, exercise-induced asthma and cold-induced asthma.
Capsaicin can induce cough by acting on TRPV1 receptors to activate sensory nerves and cause the release of sensory neuropeptides.
Asthma and COPD patients are more sensitive to capsaicin.
- The cough reflex is initiated at lower concentrations of capsaicin that do not stimulate the cough reflex in people with normal respiratory function.
- At concentration that cause cough in normal patients, the response is much stronger in respiratory disease.
- The sensory nerves seem to be hypersensitive in inflammatory airway diseases.
Sensory nerves are involved in local control, playing a potential role in exercise-induced asthma.
Water loss from the airways in exercise is thought to stimulate release of mediators and activates sensory nerves. There are increased responses to the dehydration process in asthma, causing cough and bronchoconstriction.
TRP receptors detect temperature changes, with TRPM8 detecting cold temperatures.
These are present on mast cells, the airway epithelium and sensory nerves.
These receptors may be involved in cold-induced asthma through activation of mast cells and production of mucus.
Antitussive agents
Available treatments are not very effective.
Codeine (opioid) and Dextromethorphan (non-opioid) act on µ opioid receptors in the cough centre.
They are considered the ‘gold-standard’ of cough treatment but only produce effects that are marginally better than the placebo.
- *Recent placebo-controlled studies have shown that codeine is no more effective than a placebo in suppressing cough caused by upper respiratory disorders or COPD. This contradicts older studies that did demonstrate codeine’s efficacy. Codeine’s efficacy in humans may be limited to specific situations due to the complex control mechanisms of cough.
Codeine is not readily available and should be avoided in children under 18 as it can cause addiction and respiratory depression.
Dextromethorphan should be avoided in children under 6.
Levodropropizine inhibits the release of sensory neuropeptides in vitro, producing comparable results to codeine and dextromethorphan.
Targeting sensory nerves in cough - Gefapixant
Many agents targeting sensory nerves were initially investigated for the treatment of pain and then repurposed for cough treatment.
Gefapixant is a P2X3 receptor antagonist.
P2X3 receptors are ATP-gated ion channels on sensory nerves.
ATP is released from epithelial cells in the airways in response to inflammation. It activates P2X3 receptors to stimulate sensory nerves, and hence cough.
P2X3 receptor antagonist inhibits this process.
This drug is currently in clinical application.
Targeting sensory nerves in cough - OX-314 and BW-031
In cough treatment, we want to inhibit the activity of sensory nerves without impacting other nerves.
QX-314 is a charged Na+ channel blocker
It inhibits neuronal activity, similarly to local anaesthetics.
However, anaesthetics like lidocaine are non-selective, so they inhibit sensory nerves as well as other nerves.
Therefore, anaesthetics inhibit cough, but patients also lose other reflex responses, e.g. swallowing and the gag reflex.
Na+ channel blockers act at the intracellular binding site, so they need to cross the plasma membrane.
QX-314 can’t cross the plasma membrane as it is a charged compound, but the opening of TRP channels allows it to pass through.
TRP channels are present on sensory nerves, but not other nerves, which confers this drug selectivity.
Sensory nerves are also involved in exercise and cold-induced asthma, causing both cough and bronchoconstriction, so they can be targeted for the treatment of asthma as well.
Silencing nociceptive neurons with QX-314 reduces allergic airway inflammation in asthma.
BW-031 is a new compound that’s more potent than QX-314.
QX-314 is co-applied with TRPV1 or TRPA1 agonists to cause inhibition of sensory nerves by allowing it to access the intracellular side of Na+ channels.
- TRPV1 agonists may initially worsen cough, having a similar effect to capsaicin, but sensory nerves then become desensitised.
- The initial worsening can cause coughing fits, which are not ideal.
- TRPM8 could also be activated with menthol and TRPA1 can be activated by cinnamaldehyde (found in cinnamon). These channels would also allow the entry of QX-314 without initially worsening effects.
BW-031 can also inhibit cough reflex without co-application of TRP agonists. This is because TRP channels are already activated in inflammation, so the drug can selectively target sensory nerves activated by inflammation.
BW-031 is more potent than QX-314, so the low concentrations that gain access to sensory nerves in this way are enough to inhibit Na channels.
Increasing drug concentrations decrease cough count in guinea pigs.
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BW-031 was tested in 3 models of tissue inflammation in rats and mice and a cough model induced by inhalation of dilute citric acid in guinea pigs.
BW-031 inhibited Nav1.7 and Nav1.1 channels with approximately sixfold greater potency than QX-314 when introduced inside cells.
BW-031 inhibited inflammatory pain in all three animal models tested.
BW-031 effectively reduced cough counts (by 78%–90%) when applied intratracheally under anaesthesia or by aerosol inhalation in guinea pigs with airway inflammation.
In contrast to lidocaine, BW-031 had no effect on sensory or motor function.
