lecture 20: the beta 2-adrenoceptor agonist: risk and reward

Question 1

How does the burden of disease drive frontiers?

Answer 1

• worldwide deaths attributed to:
  
  • lower respiratory infections (3rd): 3.5 million (6.1%)
  • COPD (4th): 3.3 million (5.8%)
  • trachea/broncus/lung cancer (6th): 1.4 million (2.4%)
  • tuberculosis (7th): 1.3 million (2.4%)
• DALYs (disability adjusted life years) lost worldwide:
  
  • lower respiratory infections (1st): 79 million (5.4%)
  • COPD (7th): 33 million (2.3%)
  • Tuberculosis (8th): 29 million (2.0%)
  • trachea/bronchus/lung cancer: 13 million (0.9%)
• lung health is worsening in terms of its impact

Question 2

What happens when airways are exposed to triggers?

Answer 2

• e.g. cold air or allergen in a person with atopy
• airway narrowing
• acute inflammation → typically takes a little more time to evolve than the acute spasm of the muscle
• in ongoing asthma there is also an infiltration of the airway with inflammatory cell types e.g. T cells (TH2), eosinophils → can trigger structural changes → airway remodelling
• narrows to easily and too much
• multiple parallel intersecting processes rather than a linear pathology

Question 3

What contributes to airway obstruction?

Answer 3

• airway smooth muscle shortening (narrowing of lumen)
  
  • relievers (used to relieve a perceived need of relief of spasm), controllers (used chronically to control/diminish likelihood of spasm), preventers (chronic use with the aim of reducing inflammatory cell activation)
• bronchial wall oedema (swelling - enroachment on lumen)
  
  • preventers
• mucus hypersecretion ( occlusion of lumen)
  
  • preventers

Question 4

What is the balance between mediators and airway smooth muscle?

Answer 4

• balance between constrictor and dilatory mediators on that smooth muscle
• functional antagonism
• contraction
  
  • ACh
  • HA
  • LTC4
  • LTD4
• relaxation
  
  • PGE2
  • PGI2
  • Adrenaline
  • B2-selective adrenoceptor agonists

Question 5

What is airway smooth muscle contraction?

Answer 5

• lung slice
• area as the percentage of the initial area over time
• able to induce pretty considerable narrowing

Question 6

What is the contractile mechanism?

Answer 6

• regulation of intracellular calcium
• mechanisms increasing free [calcium]
  
  • TRP channels
  • voltage operated calcium channels
  • phospholipase C/inositol trisphosphate (IP3)
  • release from intracellular stores
• mechanisms decreasing free [calcium]
  
  • plasma Ca2+ ATPase-extrusion
  • sarcoplasmic reticulum Ca2+ ATPase (SERCA)
  • uptake into internal stores
• these processes make waves

Question 7

What is airway smooth muscle relaxation?

Answer 7

• relationship between contraction and frequency of waves
• histamine causes spikes of calcium fluorescence
• formoterol causes a decreases in calcium waves → causes a concentration related decrease in spikes of calcium, decrease in frequency → relaxes
• long acting beta-agonists
• long duration of action
• given twice
• background level of bronchodilator effect
• measure of [Ca2+] vs time

Question 8

What are the molecular mechanism by which the contractile operatus is operated?

Answer 8

• g protein coupled receptor
• Gq (g protein)
• couples through to phospholipase C
• able to generate inosotil trisphosphate → triggers calcium oscillations
• calcium oscillations trigger myosin light chain kinase → phosphorylates myosin light chain (MLC) → confers on the actin/myosin filaments their ATPase activity which allows them to slide over each other and shorten → contraction
• calcium dependent process
• PLC is able to activate protein kinase C and rho kinase
• these two systems impinge on the myosin light chain phoshpatase → regulatory enzyme tending to turn off the stimulus for contraction by removing phosphate from the phosphorylated myosin light chain
• so as well as stimulation there are pathways inhibiting an inhibitory influence → reinforce the original stimulus
• PKA has influences that oppose the influences of the GPCR (targeted by B2 adrenoceptor
• contractile agonists work through Gq coupled GPCR (each agonist has its own receptor, highly selective agonists working through separate receptors)

Question 9

How do B2-adrenoceptor agonists relax airway smooth muscle?

Answer 9

• beta agonists couple through B2-adrenoceptors
• coupled to a stimulatory g-protein → Gs
• Gs activates adenolatecyclase (AC)
• increase in cAMP → activates PKA
• PKA reduces calcium wave frequency
• activates SERCA and inhibits IP3 receptors
• IP3R will open calcium stores allowing Ca2+ into the cytoplasm → inhibited by PKA
• stimulates reuptake of calcium by the SERCA
• i.e. decreases rate of release and increases rate of reuptake → reduced cytoplasmic Ca2+ → less MLCK activation → relaxation

Question 10

What are relievers?

Answer 10

• short-acting b2-adrenoceptor agonists
• short-acting:
  
  • salbutamol, terbutaline (SABA)
  • mainstay of acute bronchodilator therapy
• key features:
  
  • short acting agents: rapid (2-5 min) onset
  • B2-selective (very important)
• adverse effects:
  
  • tachychardia
  • tremor
  • hypokalemia
• other features:
  
  • variable degrees of efficacy (important??)
  • tolerance (measurable - may be important)

Question 11

What are controllers?

Answer 11

• long-acting Beta2-adrenoceptor agonists
• salmeterol - slow onset, 12 hrs duration
• formoterol - rapid onset, 12 hrs duration
• indicated for prophylaxis
• combined with inhaled glucocorticoid in single actuator
• reduce likelihood of symptoms
• no substantial anti-inflammatory action
• introduced into clinical use in late 1990s
• long duration changed nature of use

Question 12

What is the Beta2-agonist saga?

Answer 12

• 60s Isoprenaline - excess mortality, non-selective, CVS?
• late 90s Fenoterol in NZ - excess mortality, high efficacy, marketed for more severe asthma
• 90s: LABA - introduction closely monitored
• noughties - SMART - salmeterol multi-centre asthma research trial: asthma deaths increased from 0.45 to 1.98 per 1000 patient years - Black box warning and further trials
• meta-analysis suggets that there is an increase in mortality in those patients on LABAs cf those not on

Question 13

What ares plausible explanations for what might be happening with the beta agonists?

Answer 13

• chance observations (probably not)
• lack of selectivity - isoprenaline, adrenaline (doesn’t offer explanation for more recent data)
• high efficacy - fenoterol (unlikely to be the explanation, some are partial agonists)
• excessive usage - all (may be a feature in each of the studies, difficult to measure)
• inappropriate reliance on reliever/controller - inadequate anti-inflammatory treatment (sense that the disease is under control but underlying inflammation is advancing unchecked)
• beta-2 adrenoceptor dysfunction: intrinsic/acquired

Question 14

What are some surprising insights into the beta-agonist function?

Answer 14

• studies in transgenic mice
• some are beta-adrenoceptor deficient
  
  • would expect that removal would make airway obstruction worse
  • actually saw less airway obstruction
  • when the muscle is taken ex-vivo from the mouse - less contraction seen
• different set: over expression of beta-2
  
  • contrary to expectation
  • the contractile apparatus is more active with over-expression of beta-2 ADR
• an inverse beta2-agonist PROTECTS against murine “asthma”
  
  • NAD (nadolol, an inverse agonist at beta2-adrenoceptors)
  • mice b2-ADR-/-
  • less mucus production
  • non-selective
  • signalling from empty beta2-adrenoceptors facilitates asthma phenotype in mice
  • inverse agonists stop signally from ‘empty’ receptors

Question 15

What does the inverse agonist do?

Answer 15

• inverse agonist able to change the activity of the receptor
• manifests as decrease in cAMP
• system where there is some baseline stimulation you can see effects of inverse agonist
• a two state model where Ra can form spontaneously in sufficient amount to cause significant cAMP increase
• the inverse agonist decreases Ra by stabilising the pool of Ri which does not couple to AC

Question 16

Does this apply to human?

Answer 16

• answer appears to be yes

Question 17

What does nadolol do in open-label phase 1 clinical trial in mild asthma?

Answer 17

• decreases airway hyperresponsiveness
• suggests there may be ways of taking advantage of the beta-ADR that we aren’t currently aware of and that may have greater therapeutic beneftis