BM322 Block D
Lecture 1 - Pathology of COPD and Asthma
Asthma often involves cough, wheeze or chest tightness in response to a number of environmental stimuli. This is usually more pronounced in the early morning and late night. The airways of asthmatic patients are hyper-responsive to constrictor effects of a number of stimuli which include:
Exercise (non-specific)
Cold air (non-specific)
Hyperventilation (non-specific)
Chemical agents e.g. smoking (non-specific)
Allergens (specific to allergic asthma)
Aspirin (specific to hyper-responsiveness to aspirin)
Characteristics of Asthma
Asthma involves chronic inflammation which is driven by eosinophils, mast cells and neutrophils. It also involves hyper-responsiveness of smooth muscle to substances which cause contraction such as acetylcholine (parasympathetic response) and hypo-responsiveness to substances which relax the smooth muscle such as adrenaline (sympathetic response), however there is limited evidence of the dampened sympathetic response most evidence is focussed on the parasympathetic response being heightened.
There is also evidence of neuronal imbalance which causes the autonomic nervous system to be tipped towards the parasympathetic side causing more contractile responses.
Hyperplasia and hypertrophy in the smooth muscle cells is also found in asthmatic patients this means they have larger smooth muscle cells and more of them.
In healthy patients the bronchioles which are composed of contractile bronchial smooth muscle terminate at the alveoli where gas exchange occurs however in asthmatics the bronchial smooth muscle cells contact and narrow the airway lumen making it harder to breathe, the smooth muscle also goes through proliferation (hyperplasia) and cells enlarging (hypertrophy). They also become synthetic meaning that they produce inflammatory mediators that can cause contraction of the smooth muscle. Goblet cells also produce excessive mucus secretion (oedema) which cause an obstructive plug blocking the bronchiole making it harder to breathe.
Immune Prolife of Asthma
In allergic asthma allergens make it through the epithelium and dendritic cells then take up and digest the allergen presenting it to naive T cells via MHCII. This results in proliferation of the naive T cell into a Th2 cell. This results in the release of IL-4 causing an isotype shift of B cells resulting in the production of IgE. IgE is then specific to the allergen which is present and can being mast cell receptors during the sensitisation phase resulting in crosslinking which causes the production of procontractile agents leading to bronchoconstriction.
Not all asthmas are mediated by Th2, Th1 leads to the production of TNF and interferon which activates neutrophils causing long term damage.
Th17 might also be implicated in COPD (?).
Allergic Asthma
Allergic asthma has two phases. In the early phase inflammatory cells are recruited into the interstitial fluid and smooth muscle (diapedesis). These inflammatory cells then release constrictor mediators from mast cells causing bronchoconstriction. In the late phase eosinophils take up residence in the lung and release agents such as oxygen radicals, major basic proteins and PAF. Eosinophils are contractile and damaging to the epithelium so this results in a potent killing and damaging effect upon the epithelial cells and extensive damage to the epithelial lining is achieved resulting in eosinophinic disease(?).
Mast cells respond to allergens and IgE by releasing histamine, TNFalpha, LTC and D4 as well as various interleukins such as IL-1.
Eosinophils release PAF, TNFalpha, O2, eotaxin, MBP, eosinophil peroxidase, IL-4, -5, -1(beta) and -6. These all cause damage to epithelial cells as well as activating leucocytes and smooth muscle cells.
Neutrophils release ROS, neutrophil elastase and myeloperoxidase which causes damage to epithelial cells.
Most cases of asthma is Th-2 mediated with these cells causing B cells to produce IgE, this then binds to mast cell IgE receptors. Pollen causes cross-linking of receptors and causes mast cell degranulation. Eosinophil migration and activation result in chronic long term damage as well as triggering bronchoconstriction.
Remodelling
Remodelling is structural changes which are induced by inflammation, this includes a variety of gross morphological changes including epithelial damage (repair phenotype), goblet cell hyperplasia (mucus production), increased intraluminal secretions, basement membrane thickening, smooth muscle hypertrophy and hyperplasia.
This remodelling involves changes in basic lung structure which is irreversible overtime.
Due to metaplasia goblet cells will change their function overtime. More, larger smooth muscle cells result in more constriction. The presence of blood cells in the lamina propria is due to leaky capillaries.
The airway smooth muscle also changes as it has an increased number of cytokines causing more constriction. Taking muscle from chronic asthma patients shows greater contraction in response to metacholine in an isolated organ prep which indicates an intrinsic mechanism being responsible for this. There is also more stiffness in the ASM which means there is a lack of breathing induced muscle softening - expansion requires softening and overtime asthmatic airways do not soften as much. There is also an increased muscle mass within the airway smooth muscle causing increased force of contraction. They also undergo changes which allow them to release cytokines and participate in autocrine and paracrine signalling.
Muscarinic Receptors
The muscarinic M3 receptor is responsible for the contraction of the airways. this is through IP3 and calcium pathways for short term constriction however PKC is also activated which then can inhibit MLCP which allow MLC to sustain constriction after calcium is removed. Rho also performs the same function.
Potential Changes in Asthma
We have confirmed that there are higher amounts of Rho kinase due to cytokine mediated gene induction which results in more sustained contraction via the pathway above. There is also the potential for higher levels of M3 receptors and higher levels of signalling components such as PLC/Gq.
Airway Muscle Proliferation
Both hypertrophy and hyperplasia are involved in this proliferation. This is stimulated by multiple growth factors and mediators which are produced causing proliferation however these cells can also release their own mediators to act on themselves via autocrine loops or other cells via paracrine signalling, their own mediators are able to feedback on the cells causing more proliferation. Bronchial biopsies from patients are seen to proliferate more in response to growth factor which implies an intrinsic change within the cells.
Growth factor binding to receptor tyrosine kinase will cause an intrinsic tyrosine kinase activity to phosphorylate the receptor on tyrosine residues, these phosphorylation sites act as docking sights that contain SH2 domains which can attach to the phosphorylated tyrosine on the receptor.
The first protein to be recruited in GRB (growth factor receptor binder) which is a cue to activate SOS (guanine nucleotide exchange protein) which then activates Ras via exchange of GDP for GTP on the Ras protein. GTP-bound Ras then activated Raf kinase which phosphorylates and activates many more molecules of another kinase known as MEK, this phosphorylates and activates many more molecules of MAPK and leads to a substantial amplification of the original growth factor signal.
MAPK is cytoplasmic but when phosphorylated can activate gene duplication which is required for mitosis, it also stimulates protein synthesis which allows daughter cells produced from the parent cell during mitosis has sufficient protein to survive. This pathway underlies the hyperplasia seen in bronchial smooth muscle in asthmatics.
Phosphorylates MAPK can also phospholipase A2 which is an enzyme which produces arachidonate and is needed to make LTD4 which is a powerful bronchoconstrictor. This converts smooth muscle cell into a synthetic pro-inflammatory cell.
Neuronal Imbalance
There is an autonomic imbalance in asthmatics where in there is reduced adrenergic receptors on the bronchial smooth muscle making it hypo-responsive to adrenaline. There is also an increased cholinergic drive which is a consequence of inflammatory mediators in the airway lumen which activate C-fibre sensory cells in the airway epithelium which results in a reflex cholinergic drive. This then results in the release of acetylcholine that is a powerful bronchoconstrictor which underlies the hyper-responsiveness of smooth muscle in the bronchial tree.
The vasodilation mechanism is not compromised in this imbalance as the response to salbutamol is still maximal in asthma.
There are defects in cholinergic innervation. Afferent nerves such as C-fibres respond to substances such as histamine, bradykinin and prostaglandins to cause a reflex bronchoconstriction. This results in increased vagal tone which is responsible for long term effects, reflex bronchoconstriction which is responsible for the short term effects, and increased acetylcholine release leading to increased neurotransmission that is facilitates by tachykinins and thromboxanes. There is also increased post-synaptic muscarinic receptor function.
C-Fibres
An irritant or allergen will stimulate the C fibre which leads to the CNS and then activates the vagus nerve causing more release of acetylcholine onto nicotinic receptors resulting in more acetylcholine onto the smooth muscle cells. This process results in increased vagal tone. The submucosal glands are also impacted resulting in increased mucus production. C-fibres also tend to be hyperactive in inflammatory environment and can bypass the CNS/vagus nerve directly stimulating the parasympathetic ganglion. This results in reflex bronchoconstriction(?).
COPD
Chronic obstructive pulmonary disease is a chronic slowly progressive disorder which is characterised by airflow obstruction. People with this condition have decreased FEV1 and FEV1/VC ratio.
The parameters for this disease do not change markedly over several months as it is a slowly progressing disease. Most of the lung functions impairment is fixed but some reversibility can be produced by bronchodilator therapy but the damage can never be fully reversed.
There are changed in mucus gland thickness as well as air flow limitation which is due to mechanical obstruction and inflammation due to narrowed airways, loss of pulmonary elastic recoil which is due to damage and a reduction of the alveolar attachment around the walls of small airways which results in poor gas exchange. Circulatory changes are confined to advanced disease.
Smoking is the biggest known risk factor with the genetic element only accounting for between one and two percent of cases.
Chronic Bronchitis Vs Emphysema
In chronic bronchitis there is lots of mucus production. In emphysema the alveolar walls breakdown which causes there to be less surface area for gas exchange.
Cellular COPD Mechanisms
TGF beta will enhance the proliferation of fibroblasts which causes invasion of the smooth muscle tissue by fibroblasts which distorts their function resulting in fibrosis and lessened recoil.
The degree of tissue damage is modified by the extent to which repair occurs. Oxidants produced via cigarette smoking will inhibit protease inhibitors. Proteases function to get rid of damage and are short lived in normal circumstances. Protease inhibitors stop this removal when needed and if inhibited the protease activity is longer lasting.
Alpha 1 anti-trypsin inhibits trypsin-like enzymes which are damaging. It balances the activity of elastin and other destructive enzyme proteases produced during an infection and immune challenge. The deletion of the gene responsible for encoding this protein gives a higher possibility of emphysema. Only 1-2% of COPD patients have this deficiency.
Immunological Components
The epithelial/mucosal barrier is the first part of the innate response to irritants, this barrier may be reduced over time meaning infection cant be fought. There is a debatable enhancement of Th1 and Th17 signalling. CD8+ cells do direct damage via granzyme release. Damaged cells and degraded proteins function as antigens (DAMPs) and stimulate innate and adaptive systems. M2 macrophages predominate but aberrant healing??
Bronchodilator Reversibility
Lecture 2 - Pharmacology of Asthma and COPD
COPD differs from asthma in that it is not reversible with bronchodilator treatment meaning that post bronchodilator treatment the FEV1 is less than 70%. It is also more common in older patients and is less responsive to inhaled corticosteroid treatment.
The acute symptoms of COPD can be treated with short acting beta agonists, anti-cholinergic (ipratropium) or both. The persistent symptoms can be treated with long acting beta agonists or tiotropium. Severe airflow obstruction is treated with an inhaled corticosteroid. Theophylline is a bronchodilator which can be useful in treating COPD but is falling out of favour. Oxygen is also needed with progression of disease.
Long acting muscarinic agonists are used in COPD but not asthma whereas steroid are used mainly in asthma rather than COPD.
Beta 2 Adrenoreceptor Agonists
These drugs increase intracellular cAMP causing relaxation of the smooth muscle for symptomatic relief. Short acting agonists include salbutamol. Long acting agonists include salmeterol and formoterol. The long acting agonists have lipophilic groups attached which interact with the exo sites on the receptor locking the ligand onto the receptor binding site. Long acting agonists are 100x more potent meaning a lower dose is required causing less side effects.
The lipophilic group allows for longer binding to the receptor giving it a dissociation time of greater than 300 minutes. It has a 12hr action time compared to salbutamol which has a 4hr action time meaning less doses are required. There is also less desensitisation.
Beta adrenoceptor agonists activate Gs promoting the stimulation of adenylyl cyclase, this means there is promotion of cAMP formation which in turn activates PKA. PKA inhibits MLCK, promote calcium efflux and inhibt the MPK pathway by phosphorylating and inhibit Raf-1 kinase. The net effect is relaxation of the bronchial smooth muscle and therefore bronchodilation.
Desensitisation
Beta receptor adrenoceptor agonists action is relatively short lived, one factor in the short duration of action is desensitisation which is loss in response to the agonist overtime.
Desensitisation is brought about by phosphorylation of the occupied receptor by a specific receptor kinase (GRK). This leads to subsequent internalisation of the receptor meaning no receptor is present on the surface so cannot be activated.
Due to the fact that salmeterol is a partial agonist there may be less desensitisation. This means there’s less phosphorylation of GRK and less internalisation of the receptor.
Other Beta 2 Agonist Actions
Inhibition of inflammatory mediator and cytokine release as raising cAMP in most cells is anti-inflammatory this results in reductions in plasma histamine, chemotactic factors, cytokines (TNFalpha and IL-1) and eosinophil cationic protein.
Vascular permeability, there is some suggestion that this is reduced by beta adrenoceptor agonists. They also increase mucocilliary clearance by increasing ciliary beats to clear mucus.
There is also inhibition of cholinergic acetylcholine release.
Side effects of Beta2 agonist includes tachycardia, tremor, hypokalemia (low potassium due to stimulation of sodium potassium pump. Long acting beta agonists are problematic in asthma however are fine in COPD.
Anti-Muscarinics
These tend to be more common in COPD than asthma and are used in combination with beta 2 agonists.
Ipratropium is a non-selective blocker which is short acting. Due to the lack of specificity there are problems with blockage of the M2 receptor. Tiotropium is longer acting and primarily acts on M3 receptors.
The M2 receptor induces constriction. The presynaptic M2 receptor inhibits further Ach release via a negative feedback loop. By blocking both M2 and M3 receptors encourages the release of Ach due to the fact that the negative feedback is blocked leading to more acetylcholine creating more competition between ipratropium and acetylcholine for the M3 receptor making the drug less effective.
Tiotropium
This drug binds to M1, 2 and 3 with similar potency but is functionally selective for the M3 receptor as it is almost kinetically irreversible for M3 making it effectively an M3 antagonist. It is quickly removed from the other muscarinic receptors.
This drug has a slower onset than ipratropium but lasts for 24hrs so only one dose per day is required reducing the likelihood of adverse effects.
Tiotropium relaxes the airways smooth muscle acting as a bronchodilator. It also reduces mucus production via blockage of M3 on the mucosal glands. There is also a potential reduction in neutrophil migration which is responsible for a potential anti-inflammatory effect.
Steroid Drugs
Glucocorticoids dampen down many aspects of inflammation which are linked to asthma and COPD. These are widely used by inhalation (beclametasone, budesonide, fluticasone) as prophylactic therapy. The standard oral steroid is prednisolone, IV steroids are hydrocortisone and methylprednisolone.
In asthma these are used daily for the prevention of an attack rather than in response to an attack. The delivery will differ depending on the severity of the condition, oral delivery has many side effects.
These drugs are lipophilic and dimerise in the nucleus to allow their action at nuclear receptors. They bind to responsive elements of genes (GRE) and regulate transcription which causes the reduction of expression of anti-inflammatory mediators. They increase lipocortin expression. They also inhibit inflammatory genes decreasing levels of cytokines.
There is synergy between beta 2 agonists and steroids, synergy is better than additivity. There can also be potentiation where one drug makes the other response bigger.
Synergy
This involves the regulation of two different pathways which combine to give a bigger effect on a cellular response. Steroids also increase beta 2 receptor expression, increased expression levels for receptor results in more effective action. Salbutamol increases glucocorticoid receptor expression increasing half life.
Leukotriene Inhibitors
Leukotriene B4 is a neutrophil chemoattractant. Leukotriene C4 and D4 cause bronchoconstriction, increase bronchial reactivity, mucosal oedema and mucous hypersecretion. The leukotriene D4 receptor antagonists inhibit these effects - zafirlukast and montelukast.
5LOX is the enzyme involved in leukotriene synthesis, inhibition of this enzyme by zileuton inhibits the above effects. This drug is used for reducing the frequency of asthma exacerbation and has been particularly effective in aspirin induced asthma.
Zileuton has major side effects involving liver toxicity so is used less often, zafirlukast has some hepatotoxicity but less than zileuton.
Montelukast is a CysLT1 receptor antagonist. This receptor is a GCPR and is liked to the Gq pathway causing contraction of the airway smooth muscle. This drug is the third or fourth in the STEP treatment plan and is only used in severe disease states.
Xanthines
These are commonly used anti-asthmatic drugs such as Theophylline, profylline. These are also used in COPD. They word as adenosine receptor antagonists and can block the inhibitory action of adenosine upon adenylyl cyclase via its receptors and thus allow intracellular cAMP to accumulate promoting relaxation. They are also phosphodiesterase inhibitors blocking reduction in intracellular cAMP relaxes smooth muscle.
These drugs are high within the STEP programme so are not commonly used.
PDE Inhibitors
There are many PDEs which switch off cAMP and cGMP. Theophylline inhibits several PDEs so the question of whether a specific PDE inhibitor would be good in asthma or COPD treatment.
Biologics
Biologics are antibody based treatments which are directed against cytokines and leucocytes.
Tralokinumab is an anti-IL13, lbrikizumab is also anti-IL13. IL13 is produced by Th2 cells and activates eosinophils and mast cells in the allergic response. There is also mepoluzimab which is an anti-IL5 antibody and Omalizumab which is an anti-IgE antibody.
Anti-IL8 receptor antibodies have been tested in COPD but have low efficacy. Anti-TNFalpha antibodies are also not very efficacious and cause problems with cancers. Anti-IL5 and anti-IL13 have small benefits.
Protease Inhibitors
These are helpful in patients with emphysema caused by alpha 1 anti-trypsin deficiency however this is only been seen in small trials with moderate benefits. Specific protease inhibitors have some promising early results but evidence is lacking.
Lecture 3 - Emerging Therapies for Asthma and COPD
Drug discovery process
Raising cyclic AMP levels in cells of the airways is anti-inflammatory as well as relaxation since cAMP inhibits inflammation.
PKA is stimulated by cAMP, PKA inhibits NF-kB which is a transcription factor (?) for pro inflammatory cytokines. It also activates CREB a TF for anti-inflammatory cytokines.
There are many PDEs, 4, 7 and 8 are the only ones specific to cAMP.
PDEs in the Lungs
PDE4 isoforms are ubiquitously expressed. It is found in inflammatory cells, fibroblasts, pulmonary arterial smooth muscle cells, airway smooth muscle cells, epithelial cells and endothelial cells.
Studies have shown there is differential expression of PDE4 cAMP isoforms in the inflammatory cells of smokers with COPD when compared to smokers without COPD and non-smokers.
In airway smooth muscle cells Roflumilast (PDE4 inhibtor) increases cAMP in response to formoterol.
Roflumilast Biological Activity
In vitro there is decreased release of inflammatory cytokines, mediators, chemotactic factors and reduced neutrophil migration.
There is also increased apoptosis and expression of anti-inflammatory cytokines.
Ex vivo there are the same observations in cells from animal models and tissue sections.
In vivo animal studies with roflumilast demonstrated that it reduced the accumulation of neutrophils in bronchoalveolar lavage fluid from LPS treated mice. Roflumilast also prevented bleomycin induced lung infiltration of neutrophils.
There was also improved lung function in animal COPD models involving smoking, LPS and Elastin.
There is a good inhibitory effect on macrophage numbers shown in the presence of Roflumilast in the lungs of mice exposed to chronic cigarette smoke however there was no effect seen on goblet cell metaplasia.
Desired Characteristics for Roflumilast
Ideally the drug needs to be orally active showing good pharmacokinetic properties. You also need good clinical efficacy which is based upon good pharmacodynamics which is underpinned by a good ability to get to and interact with the target.
Ideally we also want few and tolerable adverse effects.
Pharmacokinetics of Roflumilast
This drug is converted by cytochrome P450, 3A4 and 1A2 to the active metabolite roflumilast N-oxide which has 90% total activity. It is almost completely absorbed after oral administration with maximum plasma concentrations within around 1 hour.
Absolute bioavailability is around 8-% as an immediate release tablet. The therapeutic dose is 500 micrograms once daily with the free drug concentration in plasma is around 1-2nM. Plasma protein binding of RF N-oxide is approximately 97%. Drug interaction studies have shown that no dose adjustment of roflumilast 500 mg was required when co-administered with erythromycin, ketoconazole, midazolam, digoxin and the antacid Maalox.
Weight Loss
Weight loss is reported in subjects treated with roflumilast and theophylline. Investigations of the mechanisms of lipolysis in human adipocytes found that PDE3B and PDE4 regulate cAMP pools that affect the activation of AMP-activated protein kinase thereby influencing lipolysis.
Rolipram a selective PDE4 inhibitor has been shown to increase plasma GLP-1 concentrations in rates, GLP-1 regulates stomach fullness so therefore patients feel fuller quicker resulting in less food consumption.
In a clinical study roflumilast once daily caused plasma glucose levels to decrease significantly more than in the placebo group.
Isoform Specific Side Effects
Gastrointestinal disturbances are class-related adverse effects for PDE4 inhibitors. Studies in knockout mice have suggested that PDE4D is the main isoform associated with emesis. Although roflumilast shows a similar specificity for PDE4D4 as for other subtypes but the gastrointestinal adverse events are less severe than with other PDE4 inhibitors.
Cilomast is 10 times more selective for PDE4D than other isozymes and this selectivity for PDE4D type nausea inducing neurons could explain the lowers tolerability seen with this compound.
Other PDE4 Inhibitors
Cilomilast shows improved lung function and reduced exacerbation rates in COPD but is associated with gastrointestinal disturbances such as emesis and nausea.
Oglemilast inhibited pulmonary cell infiltration including eosinophilia and neutrophilia.
Tetomilast is a once a day oral PDE4 inhibitor that is currently in development for COPD and ulcerative colitis.
ONO-6126 has been tested in healthy subjects and is believed to be in phase II development. ELB353 has exhibited a good efficacy profile in animal models of pulmonary neutrophilia and a further phase I trial is underway to study its safety and pharmacokinetics in healthy subjects.