Immunology Flashcards
Aulus Cornelius Celsus defined the cardinal symptoms of inflammation in his 1st century BCE book De Medicina. What are they (4)?
redness (rubor), swelling (tumor), heat (calor) and pain (dolor).
Give the causes for each of Celsus’ 4 cardinal symptoms of inflammation
- Redness – increase in local blood flow caused by vasodilator action of inflammatory mediators
- Swelling – increase in vascular permeability, proteins and fluid leak from vasculature
- Heat – as for redness
- Pain – direct action on nociceptors (sensory neurones tuned to detect noxious stimuli)
Give 3 causes of inflammation
noxious stimulation
infection
autoimmune reaction
What is the triple response of Lewis
after drawing a pointed object across the skin:
within seconds- flush
30 to 60 secs after - flare
within minutes - wheal
What causes the flush response in Lewis’ triple response to injury
within seconds of injury, a band of redness that matches the size and shape of the stimulus appears due to local release of vasodilator substances, such as histamine, from cells disturbed by the stimulus causing dilation of capillaries
What happens during flare (Triple response of Lewis )
reddening spreads to the surrounding area because of an axon reflex (neurogenic inflammation)
What happens in neurogenic inflammation
stimulated branch of a sensory nerve fibre will send an action potential to the branching point, an action potential travels orthodromically to the spinal cord giving rise to the sensation of pain and antidromically along collateral branches resulting in the release of vasodilatory substances, which causes vasodilatation of the surrounding arterioles
What happens to the inflammatory flare response if you section a central axonal branch from the injured area
sectioning of the central branch will prevent perception of the stimulus, but the flare will continue to occur until the nerve degenerates
What happens during wheal (Triple response of Lewis )
localised swelling occurs within a few minutes of the stimulus due to histamine causing increased vascular permeability
leakage of plasma from the blood to the extracellular space results in swelling due to increased tonicity of plasma compared to normal ECF
Do you have to cut someone to replicate the triple response of Lewis?
no
responses can be largely replicated by injection of histamine (effects are mediated by H1 receptors)
Noxious stimulation of a sensory neuron sends APs to CNS and also to other fibre branches. What do these branches release
what process is this?
calcitonin gene related peptide (CGRP) and substance P (SP), which are able to directly cause vasodilatation and SP is a potent activator of mast cell degranulation
neurogenic inflammation
What does production of SP in neurogenic inflammation result in
give a piece of experimental evidence of this
vasodilation and degranulation of mast cells, which leads to the local production of histamine resulting in both vasodilatation and increased vascular permeability
mast cell deficient mice display no increased vascular permeability after intradermal injection of SP
What is D
dermatographic urticaria
what is the cause
how is it treated
a condition in which the triple response is exaggerated;
largely idiopathic
generally treated with H1-receptor antagonists, but it is also treated with the monoclonal antibody omalizumab, which recognises IgE and acts to decrease mast cell activation
How does bacterial/viral/fungal infection cause inflammation (3)
directly cause inflammation through release of toxins, which cause inflammation or by lysing host cells that liberate inflammatory factors, such as ATP.
also activate the innate and adaptive immune systems
Give an example of a bacteria directly causing inflammation through toxin release
alpha-haemolysin secreted by uropathogenic Escherichia coli induces Ca2+ oscillations in cells that cause synthesis of the proinflammatory cytokines interleukins IL-6 and IL-8
What are the two key components of the host’s immune response to pathogens
1) innate response – first line of defence, non-adaptive, developed early in evolution, present in some form in many multicellular organisms
2) adaptive response – second line of defence, physical basis of immunological “memory”, emerged later in evolution, only present in vertebrates
What are PAMPs
products of bacteria, viruses, fungi etc. that they are unable to readily change to avoid detection.
What are the best studied PRRs
Toll-like receptors (TLRs)
Why are they called Toll Like Receptors
the first toll gene was identified in Drosophila melanogaster as necessary for establishing the dorsal-ventral axis, the underdeveloped ventral portion of fruit fly larvae mutant for toll evoked the exclamation, “das ist ja toll!” (“that’s amazing!”) from Christiane Nüsslein-Volhard
How many TLRs are there
what do they do upon activation
~10
activation recruit various adaptor molecules and trigger signalling cascades that result in increased expression of pro-inflammatory cytokines and interferons
On which part of the cell are TLRs expressed
. Some TLRs are expressed at the plasma membrane like TLR4, which detects lipopolysaccharide, but others are expressed intracellularly in endosomes and generally detect DNA/RNA, e.g. TLR8 detects ssRNA.
Give 3 examples of sentinel cells
macrophages, dendritic cells and mast cells
What happens when TLRs on sentinel cells are activated by PAMPs
initiates production of a variety of proinflammatory mediators, which varies depending upon the cell type activated
Name some proinflammatory mediators released from sentinel cells upon activation of TLRs from PAMPs
prostaglandins (PGE2/PGI2 both cause vasodilatation),
histamine (vasodilatation and vascular permeability)
the cytokines tumour necrosis factor alpha (TNFα) and IL-1, which induce the production of further cytokines, vascular permeability and expression of adhesion molecules on the intimal surface of postcapillary venules.
Generally how do leukocytes leave the blood vessel to reach the site of infection
Leukocytes adhere to endothelial cell adhesion molecules, which enables them to migrate out of the vascular system and attack pathogens, a process aided by pathogen generated chemotaxins and host chemokines whose expression the pathogen has induced.
Components from which 4 systems does inflammatory exudate contain
complement system, coagulation system, fibrinolytic system and kinin system
How is factor XII activated
what is the product and its role?
Factor XII becomes activated upon contacting negatively charged substances such as collagen, producing Factor XIIa, which results in the production of thrombin, plasmin and bradykinin
Give an example of the coagulation, fibrinolytic and complement system interacting
Factor XII becomes activated upon contacting negatively charged substances such as collagen, producing Factor XIIa, which results in the production of thrombin, plasmin and bradykinin. Thrombin and plasmin hydrolyse the complement protein C3 to produce active C3a and C3b
What converts fibrinogen into fibrin
thrombin
What are the roles of C3a and b
C3a stimulates mast cells,
C3b attaches to pathogens aiding their destruction by white blood cells and cleaves C5 into C5a and C5b
Give 3 roles of C5a
activates mast cells, is chemoattractant for white blood cells and also activates them
Describe the roles of C5-9
form a membrane attack complex that can attach to bacterial membranes and induce lysis (one subunit of C5b, C6, C7, C8 and 12-18 C9 subunits)
Which cells are usually first to the site of injury
neutrophils
Give the 5 steps required for neutrophils to exit the vascular system
1) PRRs detect PAMPs causing release of TNFα and IL-1 inducing expression of adhesion molecules (e.g. selectins) on the endothelium
2) Neutrophils initially tether to and are captured by the endothelium via interaction between endothelial P-selectin and neutrophil expressed ligands, such as P-selectin glycoprotein 1 (PSGL1). involves interaction between neutrophil expressed integrins eg LFA1 and ICAM1 expressed by the endothelium.
3) Transmigration across the endothelium and basement membrane takes up to ∼15 minutes and requires both integrins and further adhesion molecules such as platelet/endothelial cell adhesion molecule 1 (PECAM1)
4) Numerous chemotaxins (e.g. C5a and the bacteria derived formyl-Met-Leu-Phe, fMLF) attract the neutrophils to the bacteria invader
5) Neutrophils eliminate pathogens via intracellular and extracellular mechanisms
What are ICAM1 and LFA1
Firm arrest and adhesion involves interaction between neutrophil expressed integrins such as lymphocyte-associated antigen 1 (LFA1) and intercellular adhesion molecule 1 (ICAM1) expressed by the endothelium.
Is it faster for a neutrophil to migrate across the vascular endothelium transcellularly or paracellularly
transmigration can occur either paracellularly (between endothelial cells), or transcellularly (through endothelial cells, which takes longer ∼30 minutes).
Give some strategies used by neutrophils to eliminate pathogens (3)
phagocytosis and killing via reactive oxygen species and antibacterial proteins (e.g. cathepsin, lysozyme and defensins)
degranulation to release antibacterial proteins into the extracellular fluid
neutrophil extracellular traps (NETs), which are composed of a core DNA element alongside enzymes that immobilise pathogens, thus preventing spreading and facilitating phagocytosis, as well as likely also directly killing some pathogens
What predisposes neonates to infection
Neutrophil dysfunction is thought to be important in the development of sepsis in newborns and neonatal neutrophils fail to form NETs, which likely contributes to newborns being predisposed to infection
What TLRs/ receptors do mast cells express
what does activation of these trigger? (5)
receptors for C3a, C5a and IgE,
stimulation of these receptors results in release of histamine, heparin, leukotrienes, nerve growth factor and, unusually, pre-formed packets of cytokines.
What is 2-(1H-imidazol-4-yl)ethanamine
chemical named for what is usually called histamine, a name resulting from it being an amine occurring in tissues (histos = tissue)
Where is histamine made and found?
Histamine is formed from the amino acid histidine by histidine decarboxylase
at the cellular level histamine is found in 4 cell types: mast cells, basophils, enterochromaffin-like cells (ECL) in the gut and histaminergic neurones in the brain. In mast cells and basophils, histamine is packaged in acidic granules with a high molecular weight heparin called macroheparin
Name 3 substances that evoke histamine release from mast cells
C3a/C5a, SP, and IgE
trigger increased [Ca2+]i which leads to degranulation
How do C3a and C5a affect mast cells
C3a and C5a receptors are Gi-coupled; the βγ subunits activate PLCβ and induce intracellular Ca2+ release via IP3
this leads to degranulation and histamine release
How does SP affect mast cells
SP primarily induces mast cell degranulation, not via neurokinin 1 receptors, but rather Mas-related gene X2 (MrgX2) receptors, which in human mast cells appear to be Gq-coupled resulting in PLCβ/IP3 mediated Ca2+ release
How does IgE affect mast cells
Allergen-induced cross linking of IgE with its high affinity receptor FcεRI induces phosphorylation of the adaptor protein linker for activation of T cells (LAT) that causes activation of PLCγ/IP3 mediated Ca2+ release.
True or false
all histamine receptors are GPCRs
true There are four histamine receptors that are GPCRs (H1-4) H1= Gq/11 H2= Gs H3=Gi H4=Gi
What are the effects of H1 receptors
H1 = Gq/11 -> PLCβ -> IP3 + DAG/PKC (important in inflammation)
How do H2 receptors work
what are they important for
Gs coupled so increases AC, increasing cAMP/PKA
important for gastric secretion
How do H3 and H4 receptors work
what are they important for
both decrease AC so decrease cAMP/PKA
H3 is an important inhibitory autoreceptor in the CNS
H4 is important for chemotaxis/cytokine release
Give the 7 key effects histamine has in the body
give the receptor important for each effect
- smooth muscle contraction (H1) in the ileum, bronchioles and uterus
- blood vessel dilation, largely mediated via endothelial release of nitric oxide (H1)
- itching evoked by activation of distinct sensory nerves (pruritoceptors, H1)
- triple response of Lewis (H1)
- increased heart rate (H2)
- gastric acid secretion (H2)
- brain neurotransmission (predominantly H1-3, but also H4)
How is histamine metabolised
2 possible enzymes:
histaminase -oxidatively deaminates histamine to produce imidazole acetaldehyde,
histamine N-methyltransferase -catalyses the transfer of a methyl group on to the nitrogen of the imidazole ring to produce NT-methylhistamine
both products are inactive at histamine receptors.
What are the names of the 2 enzymes that can metabolise histamine
histaminase (also called diamine oxidase)
histamine N-methyltransferase (also called imidazole N-methyltransferase)
Give 3 allergic conditions which involve histamine
allergic rhinitis (hay fever)
urticaria (incl. dermatographism urticaria)
allergies such as nut allergy and penicillin allergy.
What responses are involved in the allergic reactions involving histamine (5)
include swelling, itchiness, nasal congestion, watery eyes and in non-human animals poor coat quality
Give 3 symptoms of severe anaphylaxis
how is it treated
throat swells
heart rate increases
blood pressure drops
rapid adrenaline treatment
What is mastocytosis
a group of conditions where too many mast cells are present leading to excessive allergic-type reactions when an attack is triggered by various factors (heat, cold, stress, infection, exercise, drugs etc.)
What causes mastocytosis in humans
Often results from a gain-of-function mutation in the receptor tyrosine kinase c-Kit/CD117 (D816V) causing enhanced mast cell proliferation and survival
What can cause mastocytosis in non humans
Mastocytosis resulting from mast cell tumours has also been described in dogs and cats, but, non-cancerous mastocytosis has also been described in dogs, but, where tested, there was no c-Kit mutation
What is the driver of symptoms in mastocytosis
histamine
What is the aim of treatment for pathophysiology associated with histamine
inhibition of mast cell degranulation
Name a mast cell stabiliser
Sodium cromoglycate
How does Sodium cromoglycate work
inhibits mast cell degranulation
MOA unclear but decreased Ca2+ influx is seen in mast cell activation in presence of sodium cromoglycate
perhaps Sodium cromoglycate inhibits an inward Cl- channel required to maintain a negative enough membrane potential to enable sustained influxes of extracellular Ca2
What can you use sodium cromoglycate to treat
mastocytosis
available as eye drops for the treatment of hay fever e.g. Opticrom and is occasionally used in the treatment of asthma
True or false
Raising [cAMP] inhibits mast cell degranulation
true
thus β2-adrenoceptor agonists (salbutamol and formoterol) and phosphodiesterase (PDE) inhibitors (theophylline) used in the treatment of asthma are partially efficacious through mast cell inhibition although their main therapeutic action is via bronchodilation
Name a monoclonal antibody used in allergic reactions
how does it work
omalizumab
recognises IgE, decreases mast cell degranulation because allergen bound IgE induces mast cell degranulation when bound to its receptor FcεRI.
Which drugs are classical antihistamines
H1-receptor antagonists
Name 2 β2-adrenoceptor agonists that are used to treat allergic reactions
name another drug which works via a similar mechanism
salbutamol
formoterol
phosphodiesterase (PDE) inhibitors (theophylline)
both types of drug work by increasing [cAMP] in mast cells to inhibit degranulation
name a first generation antihistamine
what was an issue with it
mepyramine
rapidly permeated the blood-brain barrier and caused drowsiness, and so were not suited for systemic use,
true or false
first generation antihistamines eg mepyramine are no longer in use
false
not suited for systemic use, but it is still used in topical creams for treating insect bites.
First generation drugs are also used in some cold/flu medications to aid sleeping
How did second generation antihistamines improve on first generation drugs
not cross the blood-brain barrier (terfenadine was the first)
What was the issue with the first second generation antihistamine to come to market
Terfenadine was a blockbuster drug in the treatment of hay fever ($440 million in 1996, the year before withdrawal), but there were increasing reports of people taking it developing torsade de points syndrome (also known as prolonged QT interval) and also reports of sudden cardiac death
How did terfenadine cause prolonged QT syndrome
inhibits KV11.1/hERG, which is important for repolarisation of the action potential
How is terfenadine metabolised
Terfenadine is a prodrug, metabolised by the 3A4 isoform of cytochrome p450 (CYP450) to fexofenadine, which is the active compound
Who is at enhanced risk of death from terfenadine
what else can make this drug extra dangerous
people with in conditions of diminished p450 3A4 activity (3A4 form of CYP450 metabolises terfenadine to fexofenadine, the active compound)
p450 3A4 is inhibited by many drugs, as well as bergamottin, a component of grapefruit juice
How do 3rd generation antihistamines improve on the previous 2 generations
name 2 3rd gen drugs
non-drowsy and lack cardiac side-effects
fexofenadine and loratadine
name a 1st, 2nd, and 3rd generation antihistamine
1: mepyramine
2: terfenadine
3: fexofenadine (also loratadine)
What are the main uses for fexofenadine and loratadine
treating hay fever, allergy and urticaria
Why do we think that histamine is not involved in the underlying pathology of hypersensitivity reactions in cattle
antihistamines are of little use in treating hypersensitivity reactions in cattle
How is adrenaline administered in anaphylaxis
can you use NA instead?
why might you inject a corticosteroid
administered i.m. to counteract systemic vasodilatation and reduction in tissue perfusion. also reduces bronchospasm
no: noradrenaline is such a potent vasoconstrictor it causes reflex bradycardia and is thus unsuitable
corticosteroid hydrocortisone is often co-administered for its anti-inflammatory effects
What is a potential treatment for mastocytosis in humans (other than of inhibitors of mast cell degranulation and histamine receptor antagonists)?
imatinib
only efficacious in patients not presenting with the common D816V c-Kit mutation.
Which drugs are used to control rare cases of mastocytosis in dogs
A combination of H1R and H2R antagonists
What is atopic dermatitis
how can it be treated
a relatively common allergic condition in dogs (~10%)
although H1-receptor antagonists can be used they are often inefficacious. Oclacitinib is a beneficial treatment, which works by inhibiting Janus kinases (JAK, greatest specificity for JAK1)
A canine monoclonal antibody against IL-31 has also been developed
How is oclacintinib involved in inflammation treatment
inhibits JAK
JAKs are common to many cytokine signalling pathways associated with allergic skin diseases and thus oclactinib lowers pro-inflammatory signalling and can be used as an alternative to corticosteroids and ciclosporin
Which cells are responsible for gastric acid secretion
parietal cells
What do parietal cells do
are responsible for secreting HCl into the stomach to aid digestion and kill pathogens
Where are mucous secreting cells in the stomach and what is their role
in the gastric mucosa
secrete HCO3- ions which are trapped in mucous creating a protective barrier (pH6-7)
What can disturbances in parietal and mucus secreting cells in the stomach lead to
can contribute to the pathogenesis and pain associated with peptic ulcers and gastro-oesophageal reflux disease.
What do G cells do
secrete gastrin on to ECL cells where it binds to CCK2 receptors to produce an increase in ECL [Ca2+], which causes ECL cells to release histamine.
What does histamine secreted from gastric ECL cells do
acts at Gs-coupled H2-receptors on parietal cells causing an increasing in cAMP and PKA activity. PKA phosphorylates proteins involved in trafficking of K+/H+ pumps to the apical membrane resulting in enhanced K+/H+ pump activity and thus increased H+ release
How does somatostatin affect gastric pH
it is a negative regulator of H+ release and has both direct and indirect mechanisms of inhibition
What are somatostatin’s direct and indirect methods of inhibiting H+ release into the stomach
activation of Gi-coupled SST2R on parietal cells counteracts the effects of histamine (direct inhibition),
activation of SST2R on G and ECL cells reduces gastrin and histamine release respectively (indirect inhibition).
Which antagonists are used to decrease histamine release in the stomach
CCK2 receptor antagonist (eg proglumide)
What does proglumide do
CCK2 receptor antagonist (decreases histamine secretion)
however its use in treating peptic ulcers has been surpassed
What type of drug were the first success at treating peptic ulcers
give an example
H2-antagonists
cimetidine (Tagamet)
What is a problem with cimetidine
which drug was soon competing with it
inhibit CYP450
soon had competition from ranitidine
What was the first PPI introduced
omeprazole
Describe the activation and MOA of proton pump inhibitors
what are 3 drawbacks of PPIs
converted to their active form in the acidic environment of the parietal cell and form disulphide bonds with the K+/H+ pump.
However, PPIs are broken down by H+, have a slow onset to maximal effectiveness and predominantly metabolised by CYP2C19
What metabolises PPIs predominantly
what is the issue with this
CYP2C19
exhibits significant genetic polymorphism producing extensive and poor metabolisers, especially in Asians (1-2% in Europeans vs. ~20% in Asians).
name a K+/H+ pump blocker (not omeprazole)
what is the MOA
vonoprazan
a potassium-competitive acid blocker (P-CAB)
Why is vonoprazan an improvement on omeprazole (2)
has greater stability than PPIs in H+ and CYP2C19 is less vital in its metabolism
Give methods for treating peptic ulcers (8)
PPIs/ P-CABs CCK2 inhibitors H2 antagonists antacids cholinergic blockade vagotomy eradication of Helicobacter pylori stopping NSAID use
Name an antacid
how do they help with gastric ulcer
Gaviscon
help neutralise pH in stomach
True or false
cholinergic blockage and vagotomy for peptic ulcer treatment essentially work in the same way
true
acetylcholine stimulation of muscarinic type 3 receptors (M3R) on parietal cells enhances acid release, which is then inhibited by atropine/vagotomy
both virtually obsolete
which antibiotic do you use to treat H pylori infection
clarithromycin
Does H pylori cause gastric ulcers in dogs
Helicobacter are not generally thought to be the cause although they are often present, e.g. H. heilmannii
Why do NSAIDS cause peptic ulcers
NSAIDs inhibit prostaglandin (PG) synthesis and PGE2 acts on ECL cell EP2/3R to inhibit gastric acid secretion, as well as enhancing mucin (EP4R) and bicarbonate secretion (EP1/2R).
when would you prescribe NSAIDs in combination with eg omeprazole
what can they be given instead of omeprazole
In patients at risk of developing peptic ulcers (e.g. the elderly) or those with gastric complications
misoprostol - a synthetic prostaglandin E 1 (PGE1) analogue
What is Arthrotec
a combination of the NSAID diclofenac and misoprostol that can be used in the chronic treatment of rheumatoid arthritis with misoprostol acting as a prophylactic against NSAID-induced ulceration
If you are giving a dog NSAIDs when ulcers have been induced through prior NSAID administration, which other drug should you give it
misoprostol
Etymology of bradykinin
originally identified as a slow (brady) contractor (kinin) of guinea pig ileum smooth muscle (slow being relative to tachykinins like SP)
How is bradykinin formed generally
by the action of kallikrein upon kininogens, which exist as high- and low-molecular weight forms
describe the steps involved in forming bradykinin
Hageman factor/Factor XII to become activated by contacting negatively charged surfaces, such as lipopolysaccharide, collagen or basement membrane.
Activated Hageman factor is a protease that converts plasma prekallikrein to kallikrein, which clips HMW-kininogen to bradykinin and LMW-kininogen in tissues to kallidin
How do you activate factor XII
by contacting a -ve surface
Hageman factor only contacts negatively charged surfaces, such as lipopolysaccharide, collagen or basement membrane when leaking out of leaky blood vessels during inflammation
How is bradykinin inactivated
clipped kininase II
What is kininase I activity mediated by
what does it form
via several peptidases, including serum carboxypeptidase,
results in the removal of the C-terminal arginine to form des-Arg-bradykinin, which is an agonist for bradykinin B1 receptors
Describe kininase II
a peptidyl dipeptidase that inactivates kinins by removing the two C-terminal amino acids, this enzyme is angiotensin converting enzyme (ACE)
What type of receptor are bradykinin receptors
bradykinin B1 and B2 receptors and are both Gq-coupled GPCRs
When/ where are bradykinin B1 and B2 receptors expressed and activated
B1 receptors are upregulated during inflammation through the action of cytokines, such as IL-1, and respond primarily to des-Arg-bradykinin (bradykinin itself is a very poor B1 agonist),
B2 receptors are constitutively expressed and potently activated by bradykinin and kallidin (des-Arg-bradykinin has virtually no efficacy)
What does the activation of bradykinin B1 and 2 receptors on vascular endothelium result in
causes an increase in [Ca2+], which activates cytosolic phospholipase A2 (cPLA2) resulting in prostacyclin (PGI2) production and endothelial nitric oxide synthase (eNOS) resulting in NO production
PGI2 and NO diffuse to the vascular smooth muscle cell layer and through increasing cAMP and cGMP levels respectively, mediate vasodilatation
Do ACE inhibitors only decrease vasoconstriction via ACE inhibition
no - not only reduce angiotensin II levels, thus reducing vasoconstriction, but they also cause an increase in bradykinin levels and thus enhance vasodilatation
How is bradykinin involved in pain (2)
Activation of bradykinin receptors causes activation of nociceptors and drives pain (due to increased [Ca2+]i).
Bradykinin also induces nociceptor sensitisation because activation of Gq GPCRs results in activation of PKC, which phosphorylates numerous ion channels involved in the sensation of pain.
What inhibits kallikrein
C1-esterase inhibitor (C1-INH).
What is HAE
what is the prevalence
hereditary angioedema
HAE, prevalence ~0.01%
What is the mutation in HAE
mutation in the gene encoding C1-INH causes excessive levels of bradykinin resulting in sufferers experiencing periods of severe and painful swelling
how does the cause of type I HAE differ from type II
Type I HAE results from mutations that compromise C1-INH synthesis/secretion,
whereas Type II HAE results from mutations that produce inactive C1-INH
How common is ACE inhibitor associated angioedema
what predisposes to it
0.1% but is higher (~0.5%) in African American
it is currently poorly understood what makes people susceptible to this, but there are indications that it may be linked to genetic variation in genes that regulate the immune system
How can you treat HAE
can anything else be treated in the same way
recombinant C1-INH, kallikrein inhibitors and the B2 antagonist icatibant can be used
anecdotal evidence for efficacy in treating ACE inhibitor associated angioedema.
Which drug is used to treat HAE
icatibant (B2 antagonist)
Can you exploit B1 receptors to treat pain
Plenty of research to develop B1 antagonists for inflammatory pain, but none clinically available
What are cytokines generally
a very broad group of proteins that are largely, but not exclusively, synthesised and released by cells of the immune system and generally help to coordinate the immune response
Why are mast cells unusual in terms of cytokines
why is this important pharmacologically
they contain granules containing pre-formed cytokines.
Thus, because most cells have to synthesise cytokines, drugs that inhibit cytokine transcription (e.g. corticosteroids, see later) have a major impact on immune responses
How many cytokines are there
are they all pro-inflammatory
over 100 cytokines, some of which are pro-inflammatory and some of which are anti-inflammatory.
What are the 4 main groups of cytokines
interleukins
chemokines
interferons
colony stimulating factors
What are the key pro inflammatory interleukins
what are they released from
are IL-1 (actually three cytokines: the agonists IL-1α and IL-1β and an endogenous receptor antagonist, IL-1ra) and TNFα
predominantly released from macrophages
What do IL-1 and TNFα do once released from macrophages
promote proliferation and maturation of other immune cells, as well as causing fever (IL-1).
are there any anti inflammatory interleukins
yes
which inhibit cytokine production and inhibit some T-cell responses (e.g. IL-10 and IL-1ra).
Why are ILs called interleukins
originally observed to communicate between leukocytes
Why is the name chemokines misleading
non-cytokines also act as chemoattractants and chemokines have other roles, e.g. CCL3 induces mast cell degranulation by acting at CCR1 receptors.
What does the nomenclature of chemokines relate to
the cysteine residues in the N-terminus of the peptide chain,
CXC chemokines have a single amino acid separating the two cysteines, CC chemokines have two adjacent chemokines, C chemokines only have one N-terminus cysteine and lastly, CX3C chemokines have three amino acids separating the two cysteines
these classes are also sometimes termed α, β, γ and δ.
What kind of receptor are chemokine receptors
GPCRs
What are the 3 classes of interferon and what is the role of each class
(IFN) α, β and γ, the former two have anti-viral activity and the latter has a role in induction of Th1 responses
What is the role of colony stimulating factors
stimulate the formation of maturing colonies of leukocytes and are primarily used to overcome deficits in a person’s white-blood cell count, e.g. following chemotherapy
What is NGF
Nerve growth factor
released from both macrophages and mast cells and is a potent sensitising agent, i.e. it does not excite nociceptors directly, but rather activates signalling pathways that result in a lesser stimulus causing pain
Give an example of the sensitising effect of NGF
following NGF injection, there is a >5°C lowering of the temperature required to generate pain in humans
What are the effects of NGF mediated through
mediated by the high affinity NGF receptor tropomyosin-related kinase A (TrkA).
How is NGF related to treatment of pain
in the treatment of inflammatory pain, blocking the actions of NGF is desirable and anti-NGF monoclonal antibodies such as tanezumab have been developed, but are still in clinical trials
name a monoclonal antibody against NGF
what is it hoped to treat
tanezumab
inflammatory pain
What is Annexin A1
a protein produced by many cells, which downregulates both inflammatory cell activation and mediator release, actions that are brought about by binding to the GPCR formylpeptide receptor 2 (FPR2)
What is FPR2 a receptor for
lipoxin
what are the 3 lipid mediators of inflammation
What is the main precursor for them? What is an exception to this?
leukotrienes,
platelet-activating factor
prostanoids
arachidonic acid (exception = PAF)
How are lipid mediators produced
on demand from membrane phospholipids by the action of phospholipases.
What is PAF
Platelet activating factor
Describe arachidonic acid
a 20-carbon unsaturated fatty acid containing four double bonds
AKA 5,8,11,14-eicosatetraenoic acid
eicosa refers to the 20 carbon atoms and tetraenoic to the 4 double bonds and thus lipid mediators are often referred to as eicosanoids
what is the rate limiting step for eicosanoid synthesis
liberation of arachidonic acid from membrane phospholipids and the usual one-step process involves PLA2.
What is the most important isoform of PLA2
cPLA2
which is activated by a combination of phosphorylation and Ca2+
cytosolic PLA2 (cPLA2) is activated by a combination of phosphorylation and Ca2+. Therefore, what else can stimulate it ?
can be stimulated by a plethora of substances, e.g.
bradykinin acting at B2 receptors raises [Ca2+]i
TNFα acts at TNFR1 to promote MAPK phosphorylation of cPLA2 at serine 505 and serine 727, as well raising [Ca2+]i via TNFR2.
What does the action of PLA2 result in
production of arachidonic acid (prostanoid and leukotriene precursor) and lysoglyceryl-phosphorylcholine (PAF precursor called lyso-PAF)
Which type of enzymes synthesise leukotrienes
synthesised by lipoxygenase enzymes from arachidonic acid
describe lipoxygenases
cytosolic enzymes, expressed primarily in the lungs and leukocytes
Describe how 2 lipoxygenases act of arachidonic acid
Arachidonic acid is converted by 12-lipoxygenase to produce 12-HETE (hydroxyeicosatetraenoic acid), which is a chemotaxin and 5-lipoxygenase to produce leukotriene A4 (LTA4).
Arachidonic acid is converted by 5-lipoxygenase to produce leukotriene A4 (LTA4). What further reactions occur to LTA4
converted in cells expressing the cytosolic enzyme LTA4 hydrolase to LTB4, whereas in cells expressing the membrane bound LTC4 synthase (also called glutathione S-transferase) LTA4 is converted into LTC4, which in turn is cleaved by peptidases to produce LTD4 and LTE4.
What are LTC4-LTE4 known as
the cysteinyl leukotrienes (CysLT)
What are the cysteinyl leukotrienes (CysLT)
LTC4-LTE4
What do different leukotrienes act on
what kind of receptor is each
LTB4 acts at BLT1 and BLT2 receptors,
the CysLTs act at CysLT1 and CysLT2 receptors;
BLT receptors can be either Gq- or Gi-coupled, whereas CysLT receptors are Gq-coupled.
all act on GPCRs
Different CysLTs have different efficacy at different receptors and thus CysLT metabolism has biological implications. What does this mean?
CysLT1: LTD4»_space; LTC4 > LTE4
CysLT2: LTC4 = LTD4 > LTE4.
Give some of the roles of LTB4 (3)
potent chemoattractant and activator of neutrophils and macrophages,
it upregulates neutrophil adhesion molecule expression
promotes macrophage cytokine release
Name a leukotriene found in inflammatory exudate
what does this mean
LTB4
makes LTA4 hydrolase a therapeutic target
Why was a drug targeting LTA4 hydrolase withdrawn
caused dermatitis
What 3 things do all CysLTs do
cause bronchoconstriction,
increase vascular permeability
mucous secretion
What are CysLTs released from
why is this unsurprising?
from both mast cells and eosinophils
characteristic leukocytes in airways of asthmatics and all CysLTs cause bronchoconstriction
CysLTs are released from mast cells and eosinophils to cause bronchoconstriction. How has this knowledge been used pharmacologically?
montelukast (CysLT1 receptor antagonist) is used in maintenance treatment of asthma
What are the 2 main ways to synthesise lipoxins
either 12-lipoxygenase conversion of LTA4 to LXA4
or
15-lipoxygenase conversion of arachidonic acid to 15S-HETE, which is then converted by 5-lipoxgenase to LXA4
What does LXA4 bind to
what kind of receptor is this?
what is the effect?
binds to the FPR2,
Gi-coupled
reduces neutrophil chemotaxis and degranulation, as well as acting as an antagonist of CysLT1 receptors
How is PAF formed briefly
through the action of acetyltransferase on lyso-PAF
Why is PAF misleadingly named
it has effects upon a variety of cell types and acts through GPCRs that are variously coupled
What are the effects of PAF generally (4)
increases thromboxane (TXA2) production in platelets
can be spasmogenic;
chemotactic for neutrophils
activates PLA2
Where does the name ‘prostaglandin’ originate
experiments in the 1930s showed that semen contained a lipid that could evoke uterine smooth muscle, named prostaglandin (PG) because of originating from the prostate gland
How are prostaglandins formed
Arachidonic acid is metabolised to prostaglandins by cyclooxygenases (COX)
Following the production of PGH2, it is the cell specific enzymes that are responsible for the downstream production of a particular PG or thromboxane
How does COX convert arachidonic acid to PGH2
COX catalyses two reactions:
arachidonic acid is first cyclised and oxygenated forming the endoperoxide PGG2;
the hydroperoxyl in PGG2 is then reduced to form PGH2.
Where are COX 1 and 2 found
COX-1 is constitutively expressed in many cell types, whereas COX-2 expression is induced in inflammation, although some constitutive expression does occur.
What does prostanoid refer to
PGs and TX
What does the 2 in PGH2 refer to
the number of carbon-carbon double bonds in the final structure
What happens if an acid with a different amount of C=C double bonds is used to form PGs instead of eicosatetraenoic acid (four carbon-carbon double bonds) ?
if eicosatrienoic acid (three C=C double bonds) is used in place of eicosatetraenoic acid as a substrate then the resulting PGs have one fewer double bond and are named, for example, PGE1, and if eicosapentaenoic acid (five C=C double bonds) is used as a substrate then PGs have one more double bond and are named, for example, PGE3
What are the sources of prostanoids in inflammation?
varies
, PGE2 and PGI2 tend to be produced locally by tissues and blood vessels,
PGD2 is predominantly released by mast cells and in chronic inflammation macrophages release PGE2 and TXA2
True or false
Prostanoids act at RTK
false
Prostanoids act at GPCRs and some PGs can activate multiple receptors, e.g. PGD2 can activate TP and DP receptors
Describe the coupling of different prostanoid GPCRs
- DP1, EP2, EP4 and IP receptors are Gs-coupled causing increased cAMP production
- EP1, FP and TP receptors are Gq-coupled causing an increase in [Ca2+]i
- DP2 and EP3 receptors are Gi-coupled causing decreased cAMP production
What are the predominant effects of the following prostanoids in terms of inflammation: PGD2 PGE2 PGI2 TXA2 PGF2α
- PGD2 = vasodilatation, inhibition of platelet aggregation and relaxation of GI (GI)/uterine muscle via DP1 receptors; bronchoconstriction via TP receptors
- PGE2 = bronchial/GI smooth muscle contraction (EP1), bronchodilation, vasodilatation and relaxation of GI smooth muscle (EP2), contraction of GI/uterine smooth muscle and fever (EP3), and sensitisation of nociceptors (EP4)
- PGI2 = vasodilatation and inhibition of platelet aggregation
- TXA2 = vasoconstriction, bronchoconstriction and platelet aggregation
- PGF2α = uterine contraction in humans, bronchoconstriction in cats/dogs
what is the balance in healthy blood vessels what is the balance that has to be struck with regards to platelet aggregation
how does this change if the endothelium is damaged
balance between the pro-aggregation effects of TXA2 (produced by platelets) and the anti-aggregation effects of PGI2 (produced by endothelial cells)
the balance shifts towards TXA2, platelets aggregate and the damage is sealed.
if the vascular endothelium is damaged then the balance shifts towards TXA2, platelets aggregate and the damage is sealed. How is this related to certain thromboembolic strokes
atherosclerotic plaque rupture damages the endothelium and the platelets adhere forming a clot;
part of the clot can break off and block a cerebral artery
Why do people with oily fish in their diet tend to have lower incidence of heart attacks
fish oil is high in eicosapentaenoic acids resulting in the production of PGI3 and TXA3, TXA2 is far more potent than TXA3, but PGI3 is more potent than PGI2
thus there is an overall tip in the balance towards anti-aggregation and less vasoconstriction
How do most NSAIDs work
Is it reversible?
inhibit COX by entering a hydrophobic channel on the enzyme and forming hydrogen bonds with an Arg120,
this prevents the entrance of fatty acids, like arachidonic acid, into the catalytic domain and thus PG production is stopped.
This inhibition is reversible
Why were selective COX inhibitors thought to be better than non-selective NSAIDs
COX-1 is constitutive and expressed in many tissues, whereas COX-2 is induced during inflammation.
It was thus hypothesised that drugs that only inhibited COX-2 would result in all of the benefits of COX inhibition (analgesia, decreased swelling and antipyresis etc.) without any of the side effects such as gastric bleeding
How can you make a COX inhibitor COX-2 selective
NSAIDs have to enter the hydrophobic tunnel of COX to reach Arg120 and whereas COX-1 has a narrow tunnel, COX-2 has a wider tunnel.
Consequently, drugs have been developed that have bulky sulphur-containing side groups and selectively act upon COX-2, but not COX-1 because they cannot enter the narrow, hydrophobic tunnel
Are non-selective NSAIDs common
yes
Most NSAIDs, such as ibuprofen, are non-selective because they are small and enter the hydrophobic tunnels of both COX-1 and COX-2
How can you recognise the name of a COX2 selective NSAID
generally name ends in -coxib
eg etoricoxib
Name a non selective and a COX2 selective NSAID
non-selective: ibuprofen
COX2 selective: etoricoxib
What is the most common side effect of non selective NSAIDs
Is this a trivial side effect
GI bleeding
no - accounts for approximately 5000 deaths in the UK each year
Name 3 groups NICE have identified to be at high risk of gastric bleeding
can you give these people NSAIDs
adults aged 65+, people with a history of gastric ulceration and those taking NSAIDs chronically
yes but must be co-administered with PPIs or with the PGE1 analogue misoprostol.
Why are COX2 selective NSAIDs not as safe as originally thoughout
although COX-2 is upregulated in inflammation, it is also constitutively expressed in some endothelial and vascular smooth muscle cells (e.g. in the kidney) and thus its inhibition results in decreased PGI2 production – PGI2 is vasodilatory and decreases platelet aggregation
also inhibiting COX-2 raises levels of endogenous inhibitors of eNOS, which would lead to decreased NO production. Consequently, some, but not all, COX-2 inhibitors have been associated with increased myocardial risk
Why did Merck withdraw rofecoxib
it was associated with increased myocardial risk (rofecoxib is a selective COX2 inhibitor)
Are COX2 inhibitors still in use?
Current guidance suggests that COX-2 inhibitors should be reserved for people suffering from chronic inflammation, e.g. osteoarthritis/rheumatoid arthritis, who are not thought to have a substantial cardiovascular risk and when conventional NSAIDs are thought to pose a high GI risk
NB GI problems can still occur with COX-2 inhibitors
Why might COX2 inhibitors still cause gastric ulcers
perhaps because they interfere with the healing of pre-existing ulcers
Do all NSAIDs pose a CV risk?
yes
True or false
naproxen carries a lower cardiovascular risk than other NSAIDs
false
although it was thought that the non-selective NSAID naproxen carried a lower risk, results from a recent large trial comparing naproxen, ibuprofen and a COX-2 inhibitor did not support this hypothesis (the COX-2 inhibitor did however show significantly lowered GI side effects)
How does aspirin differ from other NSAIDs
Rather than forming a hydrogen bond with Arg120, it acetylates Ser530 and irreversibly inactivates COX, an action that explains aspirin’s long-lasting actions
What type of molecule is aspirin
acetylsalicylate
Why is aspirin considered an acetylsalicylate
by donating an acetyl group to COX, salicylate is formed
What can salicylate do
reversibly inhibit COX (like most NSAIDs)
but the concentrations produced are unlikely to have a significant therapeutic effect
Explain how aspirin’s its irreversible action means that it is used at low doses for reducing the risk of platelet aggregation
platelets express COX-1 and TX synthase and are a major source of TXA2 (induces platelet aggregation and vasoconstriction), whereas endothelial cells predominantly produce PGI2 (inhibits platelet aggregation and vasodilatation);
acetylated COX-1 can be replaced by newly synthesised protein in endothelial cells, but TXA2 production by platelets is switched off for their lifetime (~10 days). Therefore, overall switch to beneficial, anti-thrombotic effects, with a lower risk of stomach ulcers (but PPIs are often co-prescribed to limit the risk.)
Is aspirin COX selective
Although weakly COX-1 selective, aspirin also acetylates COX-2
What does aspirins acetylation of COX2 result in
COX-2 no longer producing intermediates for PG and TXA2 synthesis, but instead 15R-HETE, which is the stereoisomer of 15S-HETE (product of 15-LO conversion of arachidonic acid, which is used to synthesise LXA4
5-LO then converts 15R-HETE to aspirin-triggered lipoxin – ATL, also known as 15R-epi-lipoxin A4, which has similar functions to LXA4.
What is ATL
aspirin triggered lipoxin
has similar functions to LXA2 so its production contributes to the anti-inflammatory action of aspirin
Give 4 predominant side effects of NSAIDs
Give the mechanism behind each side effect
GI bleeding (PGs inhibit gastric acid secretion and increase the release of protective mucin),
renal insufficiency and nephropathy (PGE2/PGI2 involved in maintenance of renal blood flow),
stroke/myocardial infarction (constitutive expression of COX-2 in endothelial/vascular smooth muscle cells, inhibition of which results in decreased PGI2 production, PGI2 is vasodilatory and decreases platelet aggregation),
bronchospasm (COX inhibition implicated, mechanism unclear).
What are the 2 specific side effects of aspirin
Reye’s Syndrome
Salicylism
Who gets Reye’s Syndrome
what is it characterised by
occurs almost exclusively in children (hence not recommended for use in children under the age of 16)
characterised by hepatic encephalopathy, often occurs when aspirin is taken for treating viral symptoms
Have health warnings about aspirin had any effect
yes
health warnings in the late 1980s have reduced the cases from 41/year in the 1980s to only 1 case of Reye’s Syndrome in 2002 and 3 further suspected cases up to 2009
When is salicylism seen
– essentially the result of an overdose of aspirin, often seen in children or in suicide attempts
What is the pathophysiology of salicylism
a progressive condition resulting from Kreb’s cycle inhibition and uncoupling of oxidative phosphorylation, primarily in skeletal muscle, which causes increased O2 consumption and thus increased CO2 production. CO2 stimulates peripheral and central chemoreceptors resulting in increased ventilatory rate causing respiratory alkalosis, which can be compensated for by increased renal bicarbonate excretion. However, larger doses depress the respiratory centres and CO2 accumulates, which is superimposed upon the reduced plasma bicarbonate to cause respiratory acidosis, which can combine with metabolic acidosis resulting from accumulated acidic metabolites due to disrupted carbohydrate metabolism. Fever and repeated vomiting occur, followed by dehydration, respiratory depression, coma and death
How do you treat salicylism (3)
fluids are administered, as is bicarbonate to enhance aspirin elimination,
activated charcoal adsorbs aspirin in the GI tract
in severe cases haemodialysis may be required.
What is paracetamol also known as
known as acetaminophen in the Canada, U.S., Iran and Japan
Where do the names paracetamol and acetaminophen come from
Both names are derived from a chemical name of the drug: N-acetyl-para-aminophenol and N-acetyl-para-aminophenol respectively
Why is paracetamol not really an NSAID
it is a v poor anti-inflammatory (but does have good anti-pyretic and analgesic effects)
Which enzymes does paracetamol inhibit
COX1 and 2 (but not through binding to arginine 120 or acetylation)
How does paracetamol inhibit COX enzymes
through reduction of the active site in COX enzymes required for the production of PGH2 from PGG2
How does paracetamol affect dogs
paracetamol inhibits the COX-1 splice variant COX-3 with greatest potency, and COX-3 might be preferentially expressed in the brain, however, COX-3 is likely non-functional in humans due to a shift in the reading frame producing a truncated COX protein.
How does paracetamol give analgesia
some paracetamol metabolites have also been demonstrated to have actions that contribute to analgesia
How is paracetamol eliminated usually
coagulation using hepatic enzymes
when these are saturated, oxidases act to metabolise paracetamol to NAPQI, which is usually conjugated to glutathione
What happens during paracetamol overdose
there is insufficient glutathione to eliminate paracetamol and NAPQI can oxidise the thiol groups of cellular proteins resulting in major hepato- and renal toxicity
how is NAPQI formed
when paracetamol dosage is high enough to saturate hepatic coagulation enzymes needed to eliminate the drug, oxidases (CYP2E1) metabolise paracetamol to NAPQI
NAPQI is also formed at therapeutic doses suggesting that other pathways for its formation exist
How does a paracetamol overdose present clinically
. Symptoms often do not occur until 24-48 hours post-ingestion and begin with nausea and vomiting and lead to liver failure induced death
can you reverse a paracetamol overdose
If the patient is seen soon after ingestion then prevention of liver damage can be attempted by administering substances that increase hepatic glutathione production, e.g. acetylcysteine
Name a drug given to treat paracetamol overdose
acetylcysteine
increases hepatic glutathione production
What is required to overdose on paracetamol
how has legislation tried to tackle this problem? has this been effective?
Toxicity occurs from as little as 150 mg/kg, for a 65 kg adult = 9.75 g, paracetamol often comes in packs of 16 x 500 mg = 8 g, e.g. not difficult to buy enough to overdose.
However, pre-1998, pack sizes were often 100 x 500 mg, legislation in England and Wales restricted over the counter packs in non-pharmacy premises to 16 x 500 mg,
studies show that this caused a significant reduction in paracetamol-related deaths
What increases the risk of toxicity from paracetamol
Alcohol can increase risk of toxicity because it upregulates CYP2E1, which converts paracetamol to NAPQI, fasting is also a risk, perhaps because of decreased hepatic glutathione levels.
Which enzymes converts paracetamol to NAPQI
CYP2E1
Can you give aspirin and paracetamol to cats and dogs
aspirin is not suitable for use in cats because they lack the ability to conjugate salicylate with glycine.
Paracetamol should also be avoided in dogs and especially in cats because they lack enzymes that in humans enable efficient excretion
What is ‘bute’
Phenylbutazone
NSAID used for treating horses but causes a plethora of side effects in humans (it is also abused in racehorses, which are regularly tested for bute use).
What is robenacoxib
an NSAID licensed for use in cats and dogs
What is firocoxib (species differences)
NSAID used for dogs and horses
Where does the name ‘serotonin’ come from
because it was detected in serum following clotting of the blood and caused vasoconstriction
When are high 5-HT serum levels seen
after coagulation because platelets contain 5-HT, which is released during platelet activation and aggregation and induces further platelet aggregation, as well as causing vasoconstriction in damaged blood vessels
true or false
serotonin is a vasoconstrictor
true but can also dilate
5-HT can cause both vasodilatation and vasoconstriction depending upon receptor expression
Which gut cells express high levels of 5-HT
intestinal enterochromaffin cells and in serotonergic neurones of the enteric (and central) nervous system.
How is 5-HT formed
from tryptophan through the actions of tryptophan hydroxylase (Tph) and L-aromatic acid decarboxylase (= dopa decarboxylase)
Where is 5-HT formed
from tryptophan in both serotonergic neurones and enterochromaffin cells.
What is the rate limiting step in 5-HT formation
Tph conversion of tryptophan to 5-hydroxytryptophan
What are the 2 Tph isoforms
Tph1 is primarily expressed in enterochromaffin cells and Tph2 is predominantly expressed in neurones
True or false
Platelets produce serotonin
false
platelets expresses SERT which take up 5-HT from intestinal circulation
how do platelets take up 5-HT
Platelets express a serotonin transporter (SERT) enabling platelets to become loaded with 5-HT when they pass through the intestinal circulation (platelets are not thought to synthesis their own 5-HT), which has a high 5-HT concentration
Describe the degradation of serotonin
how is it excreted and why is this useful clinically
2 steps:
1) oxidative deamination via monoamine oxidase
2) through oxidation to produce 5-hydroxyindoleacetic acid (5-HIAA)
5-HIAA is excreted in the urine and levels of urine 5-HIAA are a measure of systemic 5-HT synthesis.
What type of receptors are each of the different 5-HT receptors (7)
- 5-HT1A, B, D – F are Gi-coupled GPCRs (note that there is no 5-HT1C)
- 5-HT2A – C are Gq-coupled GPCRs
- 5-HT3 is an ionotropic receptor/ligand gated ion channel
- 5-HT4 is a Gs-coupled GPCR
- 5-HT5A is a Gi-coupled GPCR
- 5-HT6 is a Gs-coupled GPCR
- 5-HT7 is a Gs-coupled GPCR
(2A – C are Gq-coupled GPCRs; 3 is ionotropic; 1 and 5A are Gi GPCR; all the rest are Gs GPCR)
Give 2 reasons for emesis
chemicals in the intestine or blood or disturbance of the labyrinth in the ear
name 2 key components governing emesis
the vomiting centre and the chemoreceptor trigger zone (CTZ), both of which are located in the medulla
how do the vomiting centre and CTZ work together
circulating chemicals can activate the CTZ, which sends signals to the vomiting centre to produce emesis
Name a receptor important in vomiting
how is this exploited pharmacologically
Both visceral afferents that project to the CTZ and the CTZ itself express 5-HT3 receptors
inhibition of these receptors, eg by ondansetron is an effective preventative and treatment of vomiting
What is ondansetron used to treat commonly
What is its main site of action
chemotherapy-induced nausea and vomiting (CINV), which commonly results from anti-cancer drugs
its main site of action appears to be the 5-HT3 receptors in the CTZ
Now that ondansetron is off patent what can it be used to treat other than CINV
treat nausea and vomiting of most causes e.g. post-surgery, where anaesthetic drugs can lead to emesis.
What are other drugs that can be used to stop emesis (not 5-HT3 receptor blockers)
H1 receptor antagonists (effective against motion sickness and other causes of emesis)
non-selective muscarinic receptor antagonists (anti-vomiting/nausea effects via M1 receptors)
Dopamine D2 receptor antagonists (act on CTZ)
Why is metoclopramide less commonly used to treat emesis now?
can cause acute dystonia (due to effects upon dopaminergic signalling in the brain)
How is domperidone designed to be an improvement on metoclopramide?
Domperidone was designed to be more peripherally selective and is observed to produce acute dystonia less frequently
(both are Dopamine D2 receptor antagonists )
Can rodents vomit?
no
they appear to lack the brainstem component of vomiting and cannot vomit
Should you fast rodents prior to surgery
no
Rodents appear to lack the brainstem component of vomiting and cannot vomit
in fact the hypoglycaemia that can result is highly dangerous
Name 2 types of animal that cannot vomit
horses and rodents
Why can horses not vomit
because of a muscle that acts as a one-way valve to allow food down, but not up, the oesophagus – humans have this muscle too, but it is not as strong and thus we can, and do, vomit
What is a migraine
a severe headache that occurs in about 1 in 5 women and 1 in 15 men, usually beginning in young adulthood.
What are the 2 forms of migraine
with or without aura
What is aura
a progressive visual disturbance, which occurs in approximately 20% of migraineurs and is followed about 30 minutes later, by the migraine itself
Describe the presentation of the migraine itself
throbbing headache, often starting unilaterally and accompanied by nausea and vomiting, photophobia and prostration; the length of the attack is usually several hours and people may often feel “hungover” following an attack, some suggest that hangovers are a form of migraine
What is the etymology of ‘migraine’
likely derived from the Greek hemikrania, meaning pain on one side of the head
What causes migraines
Although migraine can be triggered in some sufferers by specific stimuli (e.g. particular food such as red wine or chocolate), the underlying causes are poorly understood
What are the 3 main hypotheses for the underlying cause of migraines
vascular hypothesis
brain hypothesis
inflammation hypothesis
(The brain and inflammation hypotheses are not necessarily mutually exclusive)
Describe the vascular hypothesis for the underlying cause of migraines
intracerebral vasoconstriction produces an aura and a subsequent extracerebral vasodilatation causes headache
Is there any evidence for the vascular migraine hypothesis
modern measurements have shown that although changes in cerebral blood flow occur during migraine, headache begins during vasoconstriction and not all stimuli that cause similar cerebral blood flow changes produce headache.
What is the brain migraine hypothesis
– a wave of cortical spreading depression (CSD) spreads across the brain and is associated with the aura component of migraine. CSD is a slowly propagating 2-5mm/min wave of near-complete depolarisation, which results in silencing of neuronal electrical activity for several minutes (hence depression). CSD can be triggered by elevated extracellular [K+], such as that occurring following hyperactive neuronal firing and alongside neuronal depolarisation, ionic imbalance occurs and numerous mediators including H+, glutamate, NO, arachidonic acid, and 5-HT are released. CSD implies an important role for ion channels in migraine, and many preventative drugs work on ion channels.
What is the inflammation hypothesis of the underlying cause of migraines
activation of trigeminal nociceptors that innervate the meninges and extracranial blood vessels (remember, the brain itself is not innervated by nociceptors = no pain) is the initial event in migraine resulting in pain and neurogenic inflammation through CGRP release.
Do animals get migraines
some evidence
eg a Cocker Spaniel that would display signs of fearfulness and be very quiet for 1-2 hours before onset of vocalisation, low head carriage, refusal to eat and drink and occasional vomiting. Attacks occurred 1-2 times/month, persisted for up to 3-days and were followed by 1-2 days of quietness before a return to normal behaviour.
In the case of the Cocker Spaniel who got migraines what drug was given to improve symptoms
Topiramate
opioids and NSAIDs were inefficacious
What happens electrically during a migraine
as CSD occurs there is silencing of the electroencephalograph (EEG), coupled with an increase in [K+] and neuronal depolarisation (shown by a decreased direct-current, DC)
Which mediators are released in migraines
NO, 5-HT and AA can cause activation and sensitisation of nociceptors innervating blood vessels and may even reach meningeal nociceptors due to the disruption of the BBB as a result of upregulation of matrix metalloprotease (MMP)
How might the brain and inflammation hypotheses work together to cause migraines
During migraine, as CSD occurs there is silencing of the EEG, coupled with an increase in [K+] and neuronal depolarisation
Also, mediators released, eg NO, 5-HT and AA can cause activation and sensitisation of nociceptors innervating blood vessels and may even reach meningeal nociceptors due to the disruption of the BBB as a result of upregulation of MMP activity
Activation of nociceptors results in the release of CGRP, SP and neurokinin A, which further drive inflammatory pain and is enhanced by the activation of a parasympathetic reflex involving activation of the SSN and the sphenopalatine ganglion (SPG) leading to release of VIP, NO, and ACh.
Why do we think the PNS is involved in migraines
nausea and vomiting is a common feature of migraine
How do we know 5-HT is involved in migraines (3)
During migraine: the urine levels of 5-HIAA rise
and
blood levels of 5-HT drop,
many successful anti-migraine drugs target 5-HT function
What is sumatriptan
what is it used to treat
MOA?
a 5-HT1B/D/F agonist
used to treat an acute migraine attack,
action at 5-HT1B causes vasoconstriction and 5-HT1D/F agonism inhibits nociceptor activity
What is a side effect of sumatriptan?
vasoconstriction in peripheral vascular beds is an unwanted side-effect and is contraindicated in coronary disease
What is the trouble with administering sumatriptan
poorly absorbed when taken orally, does not cross the BBB to any great extent and has short duration of action (t1/2 = 1.5 hours)
How can sumtriptan be taken
orally
nasal spray
subcut injection
sumatriptan is a 5-HT1B/D/F agonist but is contraindicated in coronary disease. What is being developed to improve on this
development of selective 5-HT1D agonists has proved therapeutically disappointing, which suggests that 5-HT1F agonists may be fruitful
Name a triptan other than sumatriptan
How do their pharmacokinetics differ
naratriptan
naratriptan has a longer duration of action, can cross BBB and has fewer cardiac side effects
Name a drug that became available in 2020 to treat migraines
Lasmiditan, a non-triptan 5-HT1F agonist
What is a benefit of lasmiditan over sumatriptan (3)
lasmiditan is 440 times more potent for 5-HT1F receptors compared to 5-HT1B/D receptors and thus does not cause vasoconstriction,
it is also far more lipophilic
readily crosses the BBB where it inhibits nociceptor activity and CGRP release
Can NSAIDs be used to treat migraine symptoms
can be used to help control acute symptoms
How does the British Association for the Study of Headaches recommend you acutely treat a migraine
treatment ladder:
starts with an NSAID (e.g. ibuprofen), sometimes with an antiemetic, such as domperidone, before moving on to triptans.
Use of painkillers should be limited to no more than 2-3 days a week or 10 days a month to prevent overuse and to allow preventative agents the opportunity to work.
Drugs can be used prophylactically for migraines. What is the principal here
to find a drug that suits the individual patient and effective drugs should generally be continued for at least 4-6 months before a trial of discontinuation
Name some drugs that can be used prophylactically for migraines (5)
propranolol amitriptyline topiramate botulinum toxin A erenumab
How does propranolol prevent migraines
who shouldn’t take this as a prophylactic
β-adrenoceptor antagonists are thought to work by preventing the extracerebral vasodilation, or by acting on the central catecholaminergic system
asthmatics
What is amitriptyline
an antidepressant used, at lower doses, as a migraine preventative
Give the MOA for amitriptyline in migraine treatment
unclear, but is thought to involve inhibition of Nav channels and inhibition of noradrenaline and serotonin uptake
What are side effects of taking amitriptyline prophylactically for migraines
can also be sedative and typically causes a dry mouth due to anticholinergic action
What is topiramate?
MOA?
an anti-epileptic repurposed as a migraine preventative
acts on numerous ion channels including voltage-gated sodium channels (inhibition) and GABA-A receptors (facilitation)
What is a common side effect of topiramate
tingling hands and feet
Botulinum toxin A can be administered as a migraine prophylactic for which type of patient?
only advisable in patients suffering from chronic migraine (defined as headaches on 15 or more days/month) and in whom standard preventative agents have failed.
How is Botulinum toxin A administered as a migraine preventative
by intramuscular injection to the head and neck
How does botulinum toxin A provide migraine relief
thought unrelated to its muscular relaxant action, but may involve decreased release of neurotransmitters from nociceptors.
Give evidence of CGRP having a role in migraine pathogenesis (2)
during an attack CGRP levels in the external jugular vein are elevated
CGRP infusion induces headaches and migraine-like attacks in migraineurs, but not in non-migraineurs.
In recent years there have been a number of randomised clinical trials investigating the efficacy of using monoclonal antibodies against CGRP or the CGRP receptor. How do these work respectively?
What else similar has been developed to treat migraines?
the former sequestering CGRP, the latter functioning as receptor antagonists
small molecule CGRP receptor antagonists
Give an example of an anti-CGRP receptor monoclonal antibody
what is it used for?
erenumab
for preventative treatment of migraine in adults and prophylaxis of migraines in adults who experience at least 4 migraines/month respectively.
What is FHM
what does it involve
Familial hemiplegic migraine, involving migraine and half body paralysis (rare)
True or false
Migraines can have sensory symptoms
true
it is common to for migraine with aura to cause one sided sensory symptoms
How do you get FHM
inherited through mutations in different genes, one of which, CACNA1C, encodes the voltage-gated Ca2+ channel 1.2 α subunit (CaV1.2).
How do mutations lead to FHM
Mutations produce variable effects in channel function and may lead to a lower threshold for CSD initiation.
how can you treat FHM
Although randomised trials have not been conducted, there is some evidence that the CaV inhibitors are efficacious in both aborting attacks and as prophylactics.
Give 2 migraine/ gene links
CACNA1C - encodes the voltage-gated Ca2+ channel 1.2 α subunit (CaV1.2) and is mutated in FHM
TRESK
What is TRESK
what is it involved in
a 2-pore domain K+ channel
plays a role in regulating the resting membrane potential and some dominant negative mutations in TRESK are associated with migraine
Why might TRESK lead to migraines
regulates resting potential and is expressed by nociceptors and loss of TRESK activity may predispose to migraine attacks
Why is TRESK potentially useful therapeutically
TRESK agonists a potential therapeutic target in the treatment/prevention of migraine.
What are the 2 phases of the adaptive immune response
the induction phase and the effector phase
How does the induction phase of the adaptive immune response begin
by an antigen being presented to a naïve CD4+ or CD8+ T cell by an APC (macrophage or dendritic cell), which has ingested a pathogen, breaks it down by proteolysis and then presents peptide fragments
What do CD4+ cells do once activated
synthesise and express IL-2 receptors and release IL-2, which acts in autocrine fashion (i.e. on itself) causing generation and proliferation of T-helper zero (Th0) cells.
Autocrine action of IL-4 causes the production of Th2 cells, which in turn activate B cells to proliferate and give rise to plasma cells that secrete antibodies and memory B cells
Name 4 elements involved in antibody mediated immunity
activation of complement,
labelling of bacteria for phagocytosis,
linkage with natural killer cells to kill antibody coated cells
activation of mast cells/basophils
What are follicular T cells involved in
B cell proliferation and differentiation.
what is iTreg and what does it act with
inducible Treg
acts in concert with nTreg cells (naturally occurring Treg) and is responsible for restraining the immune response to prevent excessive immune responses
Compare, briefly, the CD8+ and CD4+ response
Like CD4+ cells, CD8+ cells also synthesise and express IL-2 receptors and release IL-2, which acts in an autocrine manner to generate cytotoxic T cells (TC), which kill virally infected cells.
What type of immune response dominants type 1 diabetes
what about asthma
Th1 responses
Th2 response
Give 4 autoimmune reactions which involve the Th1 response
Type 1 (insulin-dependent) diabetes mellitus, multiple sclerosis, rheumatoid arthritis and graft rejection
Distinguish corticosteroids from glucocorticoids
how are mineralocorticoids
glucocorticoids is reserved for endogenous steroids that regulate glucose metabolism (e.g. cortisol), whereas corticosteroids includes both endogenous glucocorticoids and artificial steroids, such as prednisolone, that are used in therapy;
mineralocorticoids are also a subclass of corticosteroids, but do not concern us here
Where are glucocorticoids synthesised
in the adrenal cortex in response to circulating levels of adrenocorticotrophic hormone (ACTH, released from the pituitary gland)
What is ACTH release dependent upon
under the control of corticotrophin-releasing factor (CRF, release from the hypothalamus) and one function of glucocorticoids is to inhibit CRF production and thus their own systemic levels as well
Give 5 metabolic actions of corticosteroids
decreased uptake of glucose by muscle/fat (consider the impact for diabetic humans), increase gluconeogenesis, increased protein catabolism, decreased protein anabolism redistribution of fat
Give 6 anti-inflammatory actions of corticosteroids
decrease:
- influx/activity of leukocytes
- activity of monocytes
- clonal expansion of T and B cells
- pro-inflammatory cytokine production/action (e.g. IL-1, IL-2, IL-5, TNF-α, etc.)
- eicosanoid production
increase
- release of anti-inflammatory factors, e.g. IL-10, IL-1ra, lipocortin 1, annexin-A1 and IκB
What is the overall effect of corticosteroids on the immune system
to reduced activity of both the innate and adaptive immune response
what do corticosteroids bind to
the glucocorticoid receptor (GRα), which is a nuclear receptor.
How does the glucocorticoid receptor exist normally and what happens in the presence of corticosteroids
GRα is present as homomers in the cytoplasm and are bound to heat shock protein 90 (Hsp90) and other proteins.
Binding of ligand to the receptor causes dissociation of Hsp90 and enables ligand-bound GRα to form homodimers, which translocate to the nucleus and can either transactivate or transrepress a wide range of genes
How much of the genome can corticosteroids regulate
GRα can regulate 1% of the total genome
What are the 4 ways ligand-bound GRα can regulate gene expression
A, GRα binds to a positive glucocorticoid response element (GRE) within a promoter region for a gene with low transcriptional activity and activates the transcriptional machinery (TM)
B, the TM is constitutively driven by transcription factors (TF) and GRα binds to negative GREs (nGRE) causing TF displacement and repression of the TM;
C, the TM is driven at a high level by Fos/Jun TFs binding to their AP-1 regulatory site, the effect of which is reduced by GRα binding
D, the TFs P65 and P50 bind to the NFκB site promoting the TM, an effect that is prevented by prior binding of GRα.
Give 3 examples of corticosteroids having non-genomic effects
hydrocortisone rapidly inhibits neutrophil degranulation and neither GRα antagonists nor inhibitors of translation prevent this phenomenon.
IgE-mediated mast cell degranulation is rapidly inhibited by corticosteroids in a non-genomic manner, such that membrane impermeable versions of corticosteroids have identical effects to membrane permeable versions, i.e. binding to intracellular GRα is not required.
in healthy human volunteers prone to allergic rhinitis, following induction of nasal irritation with a pollen suspension, betamethasone administration markedly reduced nasal itching within 10 minutes, the speed of this response cannot be explained by genomic effects.
Give 9 side effects of corticosteroids
- opportunistic infection
- thinning of skin and impaired wound healing
- candidiasis
- osteoporosis
- hyperglycaemia – because of glucose metabolism effects (particularly problematic in diabetic humans and animals)
- muscle wasting
- stomach ulcer
- avascular necrosis of the femoral head (rare and idiosyncratic leading to a need for hip replacement)
- adverse psychiatric effects, e.g. disturbed sleep and even psychosis
Why is osteoporosis a side effect of corticosteroids
several reasons, including the reduced osteoblast/increased osteoclast activity
Why is oral thrush a symptom of corticosteroids
suppression of local anti-infective mechanisms when taken orally
name 2 drugs prescribed alongside corticosteroids
which side effects do they protect against
a bisphosphonate (bone protection)
a proton pump inhibitor (ulcer).
What can happen if corticosteroid treatment in continued long term
Cushing’s syndrome can develop
How can Cushing’s develop in the wild
from an ACTH secreting tumour
How can Cushing’s develop in dogs
Name some symptoms if this happens
in dogs receiving excessive corticosteroid treatment, or due to ACTH secreting tumours
symptoms include: pot-bellied appearance, hair loss (primarily body, not face/legs), polydipsia and polyuria.
Why should you not withdraw corticosteroids abruptly after lengthy (>1-2 weeks) therapy
can result in acute adrenal insufficiency (Addisonian crisis as the patient fails to synthesise enough steroids (and hence patients carry a “steroid card”)
withdrawal should thus be tapered to enable full HPA recovery and schedules are often patient specific.
Describe the potency and length of action of 2 corticosteroids
hydrocortisone (name given to cortisol when used as a medication) is short acting <24hrs
dexamethasone (for treating swelling and inflammation in metastatic cancer, ) is long acting with a biological half-life of >48hrs.
What is dexamethasone used to treat
for treating swelling and inflammation in metastatic cancer, as well as to Covid-19 patients requiring supplemental oxygen and/or mechanical ventilation
Give an eg of an inflammatory skin condition that can be treated using corticosteroids
eczema can be treated with a 1% hydrocortisone topical cream
name 2 brain conditions that can be treated with corticosteroids
reduce cerebral oedema in patients with brain tumours and those suffering from high altitude cerebral oedema
Would you give corticosteriods to treat Addison’s or Cushing’s disease?
used to replace physiological levels of endogenous steroids in Addison’s disease (where the adrenal glands fail to produce sufficient steroid hormone)
Give 2 causes of Addison’s disease
autoimmune destruction of the adrenal cortex and infection of the adrenal glands by tuberculosis
How many people suffer from asthma
In the UK, approximately 5.5 million people suffer from asthma, about 1 in 12 people
What disease does the underlying pathology of asthma resemble
COPD
What are 3 key characteristics of asthma
1) inflammation of the airways
2) bronchial hyper-reactivity (hypersensitivity to irritant chemicals, cold etc. and in allergic asthma, specific allergens)
3) reversible bronchoconstriction
Numerous cell types are involved in asthma pathogenesis, but what is the key leukocyte involved
Th2
give the pathophysiology of asthma
a dendritic cell in the airway submucosa takes up an allergen and presents it to a CD4+ T cell, resulting in the development of a Th0 cell, which gives rise to Th2
Th2 cells:
release IL-5 (eosinophil priming) and IL-4 and IL-13 (switch B cells/plasma cells into producing IgE)
induce IgE receptor (FcεRI) expression in mast cells and eosinophils
high circulating IgE levels also increase mast cell FcεRI expression
Why is the allergen-induced FcεRI cross-linking on mast cells in asthma bad
causes degranulation and release of histamine and CysLTs, which are both powerful bronchoconstrictors and increase vascular permeability – process is characteristic of the immediate/acute phase of an asthma attack
What do mast cells release which is involved in the late phase of asthma (5)
release IL-4, IL-5, IL-13 and TNFα, as well as a variety of chemotaxins that recruit both eosinophils and macrophages
What happens in the late phase of asthma
smooth muscle hypertrophy occurs and the eosinophils recruited to the mucosal surface release CysLTs and granule proteins such as eosinophil cationic protein and eosinophil major basic protein which both damage the epithelium resulting in airway hypersensitivity
Epithelial cells are damaged/ lost in asthma. Why is this important?
epithelial cell loss means that nociceptive C-fibres are more accessible to irritant stimuli, which is an important hypersensitivity mechanism
What are the 2 distinct ways to treat asthma
bronchodilators (predominantly for use in acute attacks)
anti-inflammatories (to limit the inflammatory components of both acute and late phases), which are usually taken as prophylactics
Which asthma patients take preventative treatment
not everyone but recommended recommended if a person experiences asthmatic symptoms/uses a bronchodilator more than twice a week, or wakes up once a week with asthmatic symptoms
What is the step wise approach to asthma treatment (4)
starts with a short-acting inhaled β2-adrenoceptor agonist used as required (e.g. salbutamol),
moving up to a regular inhaled corticosteroid (e.g. beclomethasone) to which a long-acting inhaled β2 agonist (LABA, e.g. formoterol) can be added,
then the addition of either a CysLT1 receptor antagonist (e.g. montelukast), theophylline or oral LABA (each can be tried in turn)
finally daily oral corticosteroids at the lowest dose that controls the asthma. Omalizumab can also be considered
How do β2-adrenoceptor agonists help in asthma
act upon Gs-coupled β2-adrenoceptors expressed by bronchial smooth muscle to induce relaxation and bronchodilation and β2-adrenoceptors expressed by mast cells to inhibit degranulation
How do beta2 AR agonists cause bronchodilation
increase adenylate cyclase activity = increase cAMP/PKA = myosin light chain kinase phosphorylation and inactivation
Name a short and long term β2-adrenoceptor agonist used to treat asthma
how long does each last and when is each used
salbutamol is a short-acting drug taken as needed to control symptoms, the maximum effect occurs within 30 minutes and the duration of action is 3-5 hours;
formoterol is a LABA used prophylactically and has a duration of action of 8-12 hours.
Why does formoterol have a longer action than salbutamol
because its long, lipophilic tail enables it to be incorporated into the plasma membrane, which acts as a drug reservoir, and in addition, the lipophilic tail binds to an extra binding site on the β2-adrenoceptor itself
Give 2 side effects of β2-adrenoceptor agonists for asthma treatment
tremor (excitation of β2-adrenoceptors in skeletal muscle) and tachycardia (excitation of facilitatory β2-adrenoceptors in cardiac noradrenergic nerve terminals).
Are LABAs used to treat COPD?
yes
including the “ultra” long-acting β2-adrenoceptor agonist indacaterol
What is beclomethasone
an inhaled corticosteroid, commonly used in the prophylactic treatment of asthma
Why does beclomethasone take some time to work
What does this mean they are not useful for
action is genomic
so are not useful reversing the acute bronchoconstriction during an asthma attack
How can you reduce the systemic dose of beclomethasone
what does this do
when treating asthma, can be inhaled directly
reduces side effect profile but oral candidiasis (thrush) infections can also occur
How can you aid patient compliance for asthmatics taking beclomethasone
Beclomethasone can be combined with formoterol to lessen the number of inhalers used by patients
What is the MOA of theophylline
inhibits PDE to increase [cAMP] and therefore induces bronchodilation/mast cell stabilisation by the same mechanism as β2-adrenoceptor agonists
What is theophylline used for
it is used prophylactically against asthma and not advised in treating acute attacks
Give a side effect of theophylline
commonly cardiovascular, e.g. tachycardia
Why should theophylline be used with caution in polypharmacy
is metabolised by CYP450, which can be induced/inhibited by many drugs
What is Montelukast
how does it compare to other drugs that treat the same disease
a CysLT1 receptor antagonist and can induce bronchodilation to treat asthma,
less effective than salbutamol and is used prophylactically.
Name a muscarinic agonist used to treat asthma and COPD
ipratropium (used in management of acute attacks)
What is ipratropium
give brief MOA description
a short-acting muscarinic antagonist
produces bronchodilation by inhibiting M3 receptors on the smooth muscle
true or false
You cannot use sodium cromoglycate to treat asthma
false
Sodium cromoglycate is a mast cell stabiliser and can be used prophylactically in the treatment of allergic asthma where mast cells are key players in the pathology, such as in research scientists who develop allergies to laboratory animals.
What is omalizumab?
What is it used against?
humanised monoclonal Ab against IgE (5% mouse, 95% human sequence)
recommended cases of severe, persistent allergic asthma that are refractory to treatment with corticosteroids and long-acting β2-adrenoceptor agonists.
What is the MOA of omalizumab
humanised monoclonal Ab against IgE
By binding to the Cε3 region of IgE (part of the heavy chain that binds to FcεRI), omalizumab prevents IgE binding to FcεRI, which reduces FcεRI expression and inhibits allergen induced mast cell degranulation
Why does omalizumab not activate FcεRI itself
Omalizumab is built on an IgG framework
How is Omalizumab administered
given as an injection every 2-4 weeks
Who is omalizumab recommended for
only recommended for people who frequently suffer from severe allergic asthma attacks necessitating frequent hospital attendances, where other treatments have failed
can cats get asthma
Cats can also develop pulmonary conditions showing similarities to asthma and they, like humans, can be treated with β2-adrenoceptor agonist bronchodilators like salbutamol and corticosteroids, such as fluticasone – harder to administer to a cat than a human, but inventions like the AeroKat perhaps make it (slightly) easier.
what is a pulmonary condition that horses get that is similar to asthma
how is it treated
Recurrent Airway Obstruction (RAO, or heaves), which can be treated with both bronchodilators (e.g. clenbutarol administered orally with feed) and corticosteroids (e.g. fluticasone). In contrast to treating humans, sodium cromoglycate has little efficacy, which likely reflects a more important role for neutrophils than mast cells in the pathophysiology of RAO.
What is COPD characterised by
chronic inflammation of the airways and lung tissue leading to narrowing of the airways and shortness of breath.
What accounts for the majority of cases of COPD in the a) developed world and b) the developing world
a) smoking
b) significant contribution from pollutants (e.g. smoke from cooking with wood with poorly ventilation).
what are bronchial biopsies from asthmatics characterised by
how does this differ from those from COPD patients
asthma: characterised by activated mast cells and eosinophils
COPD: enriched in neutrophils and macrophages + fibroblast proliferation (leading to small airway fibrosis)
What contributes the irreversible nature of airway narrowing in COPD
fibroblast proliferation leading to small airway fibrosis (common in COPD)
this is a stark contrast to the reversible inhibition seen in asthma
What differentiates COPD from asthma (4)
Whereas bronchial biopsies from asthmatics are characterised by activated mast cells and eosinophils, biopsies from those with COPD are enriched in neutrophils and macrophages
airway narrowing is irreversible in COPD but reversible inhibition is seen in asthma
Alveolar tissue damage is important in COPD but not asthma
whereas small airway epithelial loss is common in asthma (leading to exposure of nociceptors), epithelial cells often proliferate in COPD
Can you use SABA bronchodilators to treat COPD
not particularly effective because of the limited reversible nature of the bronchoconstriction occurring in COPD
They are not recommended for treatment on a regular basis, but can be used in acute exacerbations
Why are short acting bronchodilators not particularly effective in COPD?
the limited reversible nature of the bronchoconstriction occurring in COPD (remember, unlike in asthma that fibrosis also occurs and affects tissue elasticity).
Can LABAs be used in COPD
yes: formoterol and indacaterol are used, often in combination with long-acting muscarinic antagonists (LAMAs, e.g. tiotropium, rather than short-acting ipratropium, which is used for treating milder COPD as determined by lung function tests)
How are LABAs usually administered to COPD patients
in LABA-LAMA combination inhalers
Why is the LABA-LAMA combination effective in treating COPD
different mechanisms of action leads to additive effects: LABA = β2-adrenoceptor activation = increase AC activity = increase cAMP/PKA = myosin light chain kinase phosphorylation and inactivation
+
LAMA = M3 inhibition = prevention of PLC activation and IP3 generation
Are orally given corticosteroids used to treat COPD
they are largely ineffective, although are used in the treatment of acute exacerbations of COPD, for example when there is a coexistent infection, or where small airway inflammation is predominant
Explain the lack of efficacy of corticosteroids in treating COPD
in COPD, there is a reduction in expression and activity of histone deacetylase 2 (HDAC2): superoxide (O2-) and nitric oxide (NO) produced from cigarette smoke and activated immune cells results in the formation of peroxynitrite (ONOO-), which nitrates HDAC2 at the active site leading to its inactivation, ubiquitination and degradation.
With lowered levels of HDAC2, there is increased acetylation of GRα, which prevents it from inhibiting NFκB-driven inflammation.
What is a promising therapy for COPD
Reversing corticosteroid resistance
Can you use theophylline to treat COPD
yes: used as a sustained release preparation to treat COPD
How does theophylline work in treating COPD
bronchodilator effects (due to increased cAMP levels)
raises cAMP levels in neutrophils and other immune cells causing a decrease in chemotaxis and activation
What is the main PDE expressed in neutrophils
why is this important for COPD treatment
PDEIV (also main PDE expressed in neutrophils, T cells, ad macrophages)
neutrophils highly expressed in COPD
specific PDEIV inhibitor roflumilast is approved for treating COPD; it is used as an add on therapy to LABAs.
When is O2 therapy used in COPD patients
for those with low blood O2 levels, long-term O2 therapy is used
sometimes only necessary during exercise
NOT for smokers
Which COPD patients cannot receive O2 therapy
O2 and cigarettes do not mix: home O2 therapy should not be prescribed to patients who continue to smoke
What is ambulatory O2 therapy
when O2 therapy is only necessary during exercise
Give 3 potential COPD therapies
modulation of neutrophil chemotaxis
anti-fibrotic therapy,
inhibition of elastase activity
How are trials for drugs which modulate neutrophil chemotaxis to treat COPD going?
initial trials with CXCR2 antagonists were promising, but were discontinued when larger trials produced negative results
Why might drugs which inhibit elastase work to treat COPD
elastase elevated in COPD and may contribute to the development of emphysema
Which sex displays higher prevalence of autoimmune diseases
female
Give 2 examples of autoimmune diseases that involve the thyroid
Graves’ disease (autoantibodies activate the thyrotropin receptor causing increased thyroxine release)
Hashimoto’s disease (autoantibodies against proteins involved in thyroxine synthesis, e.g. thyroglobulin; this typically leads first to hyperthyroidism due to cell death and release of thyroxine, followed by hypothyroidism as thyroxine supplies are exhausted and not replaced)
What happens in myasthenia gravis
autoantibodies against the nicotinic receptor that prevent the effects of acetylcholine
What happens in the following:
- primary biliary cirrhosis
- rheumatoid arthritis
- primary biliary cirrhosis -> immune attack against bile ducts of the liver)
- rheumatoid arthritis -> immune attack on the synovium surrounding joints
What is the prevalence of RA
How common is severe disability following development of RA
1%
one third of whom were likely to become severely disabled due to joint damage, prior to the development of disease modifying therapies
What causes joint damage in RA
a combination of factors that are driven by activated Th1 cells:
macrophages are stimulated to release IL-1 and TNFα causing release of metalloproteinases from osteoclasts and fibroblasts that cause cartilage and bone destruction, an effect exacerbated by infiltrated inflammatory cells, e.g. neutrophils release proteases and ROS that both cause damage.
Hyperplasia of the synovium also occurs resulting in pannus formation as fibroblast-like synoviocytes proliferate and invade the joint and release proinflammatory cytokines and proteases.
True or false
RA treatment doesn’t treat underlying disease progression
some treat only the symptoms, whereas others reduce disease progression, so-called disease-modifying antirheumatic drugs (DMARDs).
How do NSAIDs affect RA
used to relieve pain and lessen inflammation but do not alter the underlying disease process
Which NSAIDs are used for RA treatment (name 4)
Diclofenac (or the misoprostol combination drug Arthrotec) has largely been superseded by naproxen
etorixcoxib
why has naproxen superseded Arthrotec for RA treatment
why is etorixcoxib also preferable
lower cardiovascular risk
COX2 selective so significant reduction in risk of GI bleeding
name an oral corticosteroid used to treat RA
when are they given as treatment
prednisolone (provides symptomatic relief and suppresses disease activity)
Due to the side-effects associated with systemic/chronic corticosteroid use, they are usually only used to treat disease flare-ups, or as a bridging therapy when waiting for a DMARD to take effect
What are the major mechanisms of corticosteroids for the treatment of RA
decreased transcription of IL-2, which is important for driving Th cell proliferation, and the decreased transcription of IL-1/TNFα
How can corticosteroids be administered in RA
orally
can also be delivered locally, i.e. intra-articular injections
Which drug is the mainstay of management of RA
is it used alone?
methotrexate
can be used alone or in combination with other DMARDs
How does methotrexate work in RA treatment
folic acid inhibition lowering the production of tetrahydrofolate that is required for purine nucleotide and thymidylate synthesis and thus is anti-proliferative, reducing the immune response
How does methotrexate provide folic acid inhibition
inhibition of dihydrofolate reductase and competition with folic acid transport into cells
How long does it take for the effects of methotrexate in RA treatment
How is it usually administered
3–12 weeks
orally to be taken once a week, but can also be administered by weekly subcutaneous injection; weekly administration is important (NOT daily)
give 2 side effects of methotrexate
how can these be limited
bone marrow suppression and GI disturbance
weekly folic acid supplement
What is a common but deadly prescribing error seen with methotraxate
incorrect daily administration of methotrexate is a recognised prescribing error that can prove fatal due to the profound effects on bone marrow suppression and impairment of immune system function
What is a popular first choice DMARD for IBS (not methotrexate)
Sulfasalazine
How is Sulfasalazine administered
how is it metabolised
what is a likely MOA
taken orally daily
broken down by colonic bacteria to produce sulfapyridine (a sulphonamide) and 5-aminosalicylic acid (a salicylate) and one likely MOA is that the 5-aminosalicylic acid scavenges ROS released from neutrophils
When do the therapeutic benefits of sulfasalazine seen
not until after 3 months
What are unwanted effects of sulfasalazine
GI disturbances, skin rashes, bone marrow suppression hepatotoxicity tears and contact lenses can be stained yellow urine may appear orange
What was hydroxychloroquine developed to treat
malaria (also now used as a DMARD)
MOA of hydroxychloroquine
As a lipophilic weak base, hydroxychloroquine accumulates in cytoplasmic acidic vesicles, which can reduce antigen presentation by macrophages and reactive oxide species generation in neutrophils
What are some side effects of hydroxychloroquine (6)
ocular toxicity, GI upset, skin reactions, seizures, myopathy, psychiatric disturbance.
What does leflunomide do
inhibits dihydroorotate dehydrogenase, a key enzyme in pyrimidine synthesis, and thus it inhibits synthesis of DNA and RNA, with its main effect being on rapidly dividing cells such as B and T cells.
What can leflunomide be used to treat
side effects?
RA
Adverse effects include GI disturbance, bone marrow suppression and hepatotoxicity.
Which drugs are reserved for RA patients who have failed conventional DMARDS (5)
what are these often more effective with
biological DMARDS (Anti TNFα therapies, Anti-IL-6 therapy, Anti-B cell therapy, Anti-T cell therapy, Anti-IL-1 therapy)
as combination treatments with methotrexate
Name 3 drugs used in anti TNFα therapies
Etanercept
Infliximab
adalimumab
What is etanercept
a fusion protein of the soluble TNFα receptor and the Fc portion of IgG1, which acts to mop up the high levels of TNFα that occur in RA
What are Infliximab and adalimumab
monoclonal anti-TNFα antibodies (mAb, chimeric and human respectively) that, like etanercept, sequester TNFα.
What are adverse effects of anti TNFα therapies (4)
allergic reactions, bone marrow suppression,
increased susceptibility to infections (particularly tuberculosis and hepatitis B reactivation)
MS
Give an unexpected side effect of anti TNFα therapies
several cases of multiple sclerosis have been reported in patients following anti-TNFα treatment for inflammatory arthritis
Name a drug used in anti IL-6 therapy
what does it target
Tocilizumab (a mAb)
targets the IL-6 receptor, inhibiting the action of IL-6.
Why can tocilizumab be used to treat RA
IL-6 is a pro-inflammatory cytokine and, like TNFα and IL-1 is upregulated in RA. IL-6 also drives the production of c-reactive protein (CRP), which is commonly measured in blood tests, and so tocilizumab tends to be targeted towards patients with significantly elevated levels of CRP.
Name a drug used in anti-B cell therapy
rituximab (a mAb)
What does rituximab target
Name a disease that it can be used to treat
CD20 that is primarily expressed by B cells,
can be used in the treatment of RA, its therapeutic effect being through B cell destruction.
What risks are associated with rituximab
increased risk of infection (like all DMARDs)
especially, an increased risk of a potentially fatal brain infection called progressive multifocal leukoencephalopathy, which is caused by reactivation of the John Cunningham (JC) virus.
Name a drug used in anti-T cell therapy
describe it
abatacept
a synthetic fusion protein of the costimulatory cytotoxic T-lymphocyte protein 4 (CTLA-4) and the Fc fragment of human IgG1
Give MOA of abatacept
binds to CD80 and CD86 on antigen presenting cells, so interferes with the ability of antigen presenting cells to activate T cells
Name a drug used in anti-IL1 therapy
describe the drug
anakinra
a recombinant version of the IL-1 receptor antagonist, which binds to the IL-1R with a higher affinity than IL-1 itself, but does not initiate receptor signalling
When do you use anakinra to treat RA
On the balance of clinical benefits and cost-effectiveness, it is not recommended for treatment of RA in the UK, but is used in other countries
Is methotrexate used in animals
although usually reserved for treating certain forms of cancer methotrexate can also be used to treat dogs with arthritis.
How do you treat RA in horses and dogs
NSAIDs and corticosteroids
How do the different classes of biological DMARD compare (4)
All biological DMARDs are given by injection, either intravenous or subcutaneous,
all are expensive.
They are all associated with an increased risk of infections.
Each class has similar efficacy, but failure to respond to one class does not determine response to another class, so they tend to be tried serially
What is a major drawback of the therapeutic avenue of immunosuppression to prevent eg graft rejection
the immune system is compromised and thus less able to respond to infection
Name 3 steroid sparing agents
azathioprine
methotrexate
mycophenolic acid.
How do corticosteroids exert their immunosuppressive effects
via decreasing the transcription of a variety of cytokines, including those that drive Th cell proliferation and those that drive inflammatory responses, (e.g. IL-1 and TNFα), while at the same time upregulating anti-inflammatory factors such as IL-1ra
Which cytokine that drives Th0 proliferation is inhibited by corticosteroids
what does it cause
IL-2 which causes generation and proliferation of Th0 cells and proliferation of Th0 cells into Th1 cells
Name an anti inflammatory factor that is upregulated by corticosteroids
IL-1ra
How does azathioprine work
is a prodrug for 6-mercaptopurine (6-MP), which is converted to thioinosic monophosphate (TIMP), which is converted by two different enzymes to produce two products that inhibit DNA function:
Thiopurine methyltransferase (TPMT) converts TIMP to 6-methyl-TIMP (MeTIMP), which inhibits de novo purine synthesis
and
inosine monophosphate dehydrogenase (IMPDH) converts TIMP to 6-thioguanosine nucleotides (6-TGN), which are incorporated into cellular nucleic acids resulting in inhibition of nucleotide and protein synthesis
How is 6-MP converted to TIMP
by hypoxanthine phosphoribosyl transferase (HPRT)
What is the overall effect of azathioprine
why is this useful
inhibits cellular proliferation
has a greater effect upon rapidly proliferating cells, such as T cells and B cells when activated, and thus azathioprine inhibits T cell and B cell proliferation making it a useful drug in preventing transplant rejection and autoimmunity
Name 3 conditions you can use azathioprine to treat
IBS
RA
graft rejection
What are the side effects of azathioprine
effects include GI upset, hepatotoxicity and bone marrow depression
(you can also get accumulation of cytotoxic 6-thioguanine nucleotide analogues leading to toxicity)
How common is accumulation of cytotoxic 6-thioguanine nucleotide analogues as a side effect of azathioprine
why is this
how can this be prevented
10%
TPMT levels vary, 10% of people having very low activity and thus accumulation of cytotoxic 6-thioguanine nucleotide analogues leading to toxicity
by checking TPMT levels, or close monitoring of blood counts/liver function on starting treatment
Can azathioprine be used in dogs
yes as an immunosuppressant
True or false
methotrexate is a folic acid analogue
true
targets rapidly proliferating cells, such as those of the immune system. It is used in the treatment of many autoimmune conditions such as RA
What is mycophenolic acid
derived from the fungus Penicillium stoloniferum, which inhibits IMPDH, an enzyme crucial for de novo guanosine synthesis (so has greatest effect on rapidly dividing B and T cells)
What is mycophenolic acid used for
used to diminish transplant rejection but also used in autoimmune diseases such as myasthenia gravis.
How is cyclophosphamide activated
a prodrug, being converted by hepatic P450 enzymes first to aldophosphamide and then the active phosphoramide mustard
(alkylating agent)
MOA of active phosphoramide mustard
side effects?
has two alkylating groups and cross links guanine in DNA via N7 groups
bischloroethylamine undergoes cyclisation, releasing Cl- and forms an unstable immonium cation, the strained ring opens to form a reactive cabonium ion, which reacts with guanine’s N7 to produce 7-alkylguanine.
DNA replication is thus defective because 7-alkylguanine pairs with thymine producing substitution of A-T for G-C, it can also cause guanine excision and chain breakage.
immunosuppressive effect is due to killing of dividing T and B cells, but this occurs alongside side effects: bone marrow depression, potential infertility, bladder irritation and cancer
Why can cyclophosphamide lead to cancer
is this seen anywhere else
its metabolite acrolein activates TRPA1 expressed by bladder innervating nociceptors,
acrolein is also present in cigarette smoke and some forms of tear gas
What is cyclophosphamide used to treat
used to treat refractory autoimmune conditions and some cancers
What is CsA
Ciclosporin A
an immunosuppressive isolated from the fungus Tolypocladium inflatum
Describe the the structure of CsA
What is its main action
a cyclic, 11 amino acid peptide
predominant action is to prevent T-cell proliferation via suppression of IL-2 synthesis.
What normally happens within the T cell when an antigen interacts with the TCR?
How does this differ when CsA is present
increase in [Ca2+]i that stimulates the activity of a serine-threonine phosphatase called calcineurin (CaN), which then dephosphorylates the nuclear factor of activated T-cells (NF-AT) enabling its translocation to the nucleus and consequent upregulation of IL-2 transcription
CsA binds to a cytoplasmic protein called cyclophilin (CpN), which is member of the immunophilin group of proteins. The CsA-CpN complex binds to and inhibits CaN, which thus prevents NF-AT dephosphorylation and it is retained in the cytoplasm preventing IL-2 upregulation and is thus used in suppressing the rejection of transplanted organs
What is CsA used to treat in dogs
atopic dermatitis
What is another name for Tacrolimus
What is this drug
FK506
macrolide antibiotic produced by Streptomyces tsukubaensis, which binds to an immunophilin called FKBP
What is the importance of FK506 binding to FKBP
the complex inhibits CaN (similar to the action of the CsA-CpN complex)
thus thus NF-AT is retained in the cytoplasm and IL-2 synthesis is inhibited
What are tacrolimus and CsA used to treat
transplant rejection
What is basiliximab t
a monoclonal antibody against the CD25 alpha subunit of the IL-2 receptor and thus functions as an IL-2 receptor antagonist
Why is basiliximab effective in preventing transplant rejection
it is an IL-2 receptor antagonist. IL-2 receptors are upregulated on activated T-cells and B-cells so basiliximab decreases the incidence and severity of acute rejection following transplantation.
Name a immunosuppressive drug that works in a similar way to abatacept
belatacept
Describe the structure of belatacept
fusion protein of the extracellular domain of cytotoxic T-lymphocyte protein 4 (CTLA-4) and the Fc fragment of human IgG1
T cells require co-stimulation for activation. What does this involve?
Which drug exploits this
2 signals: 1) interaction between the major histocompatibility complex (MHC) of the APC with the T cell receptor
and
2) interaction between CD80/86 ligands of the APC with CD28 of the T cell
Belatacept - acts to bind CD80/86 and prevent T cell costimulation
What does the CTLA-4 of a T Cell do
Why is the CTLA-4 domain of belatacept special
its activation by CD80/86 sends an inhibitory signal to the T cell
has 2 amino acid variations that confer greater affinity for, and slower dissociation from, CD80/86
When is belatacept used
used in combination with other drugs to prevent graft transplant rejection
What can happen if you stimulate CD28 on T cells alone
How was this information used pharmacologically
can cause activation and proliferation of regulatory T cells, which are involved in preventing autoimmune reactions
an anti-CD28 agonist monoclonal antibody TGN1412 was trialled following promising results from non-human animal tests, but was abruptly ended after causing a cytokine storm in humans due to its activating effects being non-selective, i.e. in humans at the dose used pro-inflammatory memory T cells were activated alongside regulatory T cells
Does TGN1412 have a future?
maybe but must be administered at lower doses than in original trial to enable selective activation of Treg cells
Give other name of rapamycin
what is this drug
sirolimus
a macrolide antibiotic produced by Streptomyces hygroscopicus, which like tacrolimus binds to FKBP and inhibits mTOR
How does sirolimus differ from tacrolimus
both bind to FKBP but sirolimus-FKBP inhibits mTOR whereas tacrolimus-FKBP inhibits CaN
What is mTOR
what is the main target of sirolimus
mammalian target of rapamycin (mTOR), which is a serine/threonine kinase involved in cell cycle progression and protein synthesis
mTORC1 (mTOR complex 1)
What is the overall MOA of sirolimus
binds FKBP, forming a complex which inhibits mTOR, inhibiting mTORC1 especially.
this leads to decreased T cell activation and proliferation and a decreased immune response
What are the uses of sirolimus (2)
prevent transplant rejection
used to coat coronary stents to prevent restenosis (thanks to its antiproliferative effects)
How do you create a polyclonal antibody
host organism can be injected with an antigen (e.g. inactivated bacterial toxin), serum extracted from the host would contain antibodies against the antigen, but they would be a mixture of antibodies because numerous plasma cells react to a particular antigen and produce different antibodies
What are 2 drawbacks of polyclonal antobodies
Each serum generated in this manner will be of variable potency and there is only so much serum that can be collected
What is the limit of collecting serum for polyclonal antibodies
for generating antibodies for the lab, one standard protocol involves injecting two rabbits with antigen and collecting a total of 75 ml/rabbit over 70 days
How did Milstein and Köhler describe making a mAb
fusing single mouse B-cells with immortalised tumour cells to produce a hybridoma that could grow indefinitely to produce a single antibody
2 advantages of mAbs over polyclonal Abs
mAbs have known potency and there is no limitation to the amount that can be produced.
Give the full method for creating a mAb (3)
Mice are firstly injected with an antigen and antibody producing B cells are isolated from the spleen, these are then mixed with myeloma cells (HPRT-ve) in polyethylene glycol to produce hybridomas;
hybridomas are selected by growing in a medium containing a dihydrofolate reductase inhibitor, which kills off unfused myeloma cells, whereas hybridomas can create new nucleotides from supplements in the medium because they express B cell HPRT, unfused B cells have a short life and die off.
B cells are then screened for specificity (e.g. using an ELISA) and then using limiting dilution individual cells/clones are isolated, which can be propagated and then mAb collected
Describe the 2 key regions of an antibody
2 key domains:
Fc region - reacts with cell surface Fc receptors that are expressed by neutrophils (FcγRI, respiratory burst and phagocytosis), macrophages (FcγRII, phagocytosis) and natural killer cells (FcγRIII, antibody-dependent cell-mediated cytoxicity, ADCC)
Fab- contains the variable and hypervariable regions (also known as the complementarity determining regions, CDR), which bind the antigen in question
Why were 1st gen mAbs problematic (3)
a mouse injected with, for example, human TNFα will make mouse antibodies (which can be isolated as mAbs using hybridoma technology) and thus when injected into humans they were observed to produce an immune response in over half of recipients because of the generation of human anti-mouse antibodies
relatively low circulatory half-life
unable to activate human complement
What is Muromonab-CD3
murine mAb that targets CD3
was the first mAb to be approved for clinical use in humans to reduce acute transplant rejection
Go through the stages of mAb development and give an example of each
Using recombinant DNA technology, the first strategy was to create chimeric mAbs that contained the human Fc region, but mouse variable region (e.g. infliximab).
Next, humanised mAbs were developed where only the CDR was of mouse origin and these produce very little immune response (e.g. omalizumab, alemtuzumab and rituximab)
What is the benefit of chimeric and humanised mAbs over murine ones
In both chimeric and humanised mAbs, the presence of the human Fc region lengthens the plasma half-life because they can bind to neonatal Fc receptor (FcRn) on endothelial cells and are recycled.
How can human mAbs be made (2)
name one that is in clinical use
either by using transgenic mice in which the mouse immunoglobulin genes are swapped with the human equivalents, or through phage display
adalimumab
How are mAbs administered
By injection every 2 weeks (although this differs greatly e.g., infliximab is administered every 2 weeks and others once per month)
Are there still risks with taking mAb medication
Due to their high specificity, there are few off-target effects, but mAbs that target functioning of the immune system can precipitate latent disease or encourage opportunistic infections.
What does alemtuzumab do
what is it used to treat
binds to CD52, an antigen on mature lymphocytes, but not the stem cells from which they derive, and targets them for destruction
used in treating chronic lymphocytic leukaemia, cutaneous T cell lymphoma, T cell lymphoma and multiple sclerosis
What do each of the mAb suffixes mean
- ximab = chimeric
-axomab = rat/mouse hybrid
-zumab = humanised
–umab = human
True or false
mAbs are only used in human medicine
false (but used to be true)
Nexvet used a process called PETization to make canine, feline and equine mAbs and was bought out by Zoetis, who have marketed an anti-IL-31 mAb for treating dermatitis in dogs. Publications suggest that anti-NGF mAbs are in late-stage development for treating chronic pain such as that experienced in feline/canine osteoarthritis.
What do bi-specific Abs do
involve stitching together two halves of different antibodies, the idea is that one antibody binds the target cell and the other binds a cytotoxic cell, i.e. the killing machinery is localised to the target
Give an example of a bi-specific mAb
describe
catumaxomab
a rat/mouse hybrid mAb that binds epithelial cell adhesion molecule and CD3 on tumour cells and T cells respectively leading to the destruction of the cancer cell
What is catumaxomab licensed to treat
ascite in cancer patients
Why has catumaxomab been termed trifunctional
the Fc portion can bind the Fc receptors on macrophages etc
Give a possivle option for development of a bi-specific mAb to treat inflammation
one that can sequester two molecules simultaneously, e.g. TNFα and IL-1.
How can you engineer improvement of Ab-NK cell coupling
most antibodies and serum IgG are fucosylated, but antibodies engineered to be afucosylated have a higher affinity for the FcγRIII on NK cells and overcome competition with serum IgG thus increasing the window of time for ADCC to occur.
How does fucosylation of Fc affect its receptor
Fucosylated Fc causes displacement of the oligosaccharide tree connected to Asn162 of FcγRIII, which leads to an increased distance of the carbohydrate-carbohydrate contact between Fc and FcγRIII and thus reduced bond strength that explains the reduced affinity of fucosylated Abs
True or false
fucosylated Fc Abs can outcompete afucosylated, serum IgG
false
afucosylated Fc Abs can outcompete fucosylated, serum IgG
Fucosylated Fc causes displacement of the oligosaccharide tree connected to Asn162 of FcγRIII, which leads to an increased distance of the carbohydrate-carbohydrate contact between Fc and FcγRIII and thus reduced bond strength
What is the other name for Herceptin
what is Herceptin used to treat
Is this an effective treatment
trastuzumab
used to treat HER2 positive breast cancers
yes but resistance often arises
How can Herceptin treatment of HER2 positive breast cancer be improved (give details of MOA)
Trastuzumab emtansine (T-DM1) is a combination of trastuzumab (T) with a derivative of the antimicrotuble compound maytansine (DM1).
Binding of T-DM1 to HER2 causes internalisation, lysosomal degradation and release of DM1, which can bind to tubulin and prevent polymerisation causing cytotoxicity;
T-DM1 also retains the basic actions of trastuzumab: disruption of HER2 signalling and targeting tumour cells for ADCC
Why would pharmacologists mutate the Fc region of some mAb
(think pH)
give an example
improved pharmacokinetics by mutation of the Fc region to improve binding to FcRn at low pH so that more is recycled out of cells and not broken down by intracellular degradation machinery.
Fc portion of IgG binds FcRn with high affinity at acidic pH (e.g. in endosome), but lower affinity at physiological pH, increasing this affinity further would increase the circulation time.
What is the problem with mAbs being large proteins
diffusion into tissues is slow and access to intracellular targets is problematic
mAbs are large proteins and thus diffusion into tissues is slow and access to intracellular targets is problematic. How can we overcome this? (3)
shrink the peptide scaffold that displays the antigen binding loops
create bicyclic peptides, which due to their rigid conformation can bind with high affinity and specificity to protein targets, but are more resistant to serum proteases than linear peptides
creation of nanobodies
What are nanobodies
the isolated variable heavy chains, containing the CDRs, from Abs produced by camelidae and certain cartilaginous fish - they are single domain abs with low Mr
What are 2 advantages of using nanobodies
disadvantage?
+larger loops in camelidae CDRs enables greater antigen-interacting surface than heavy chain CDRs from standard IgG Abs.
+a low molecular weight (~12-15 kDa vs. ~150 kDa of conventional mAbs) means they are likely to have better tissue permeability and tissue penetration
- also means they have a lower plasma half-life
What is osteoarthritis (OA)
a degenerative joint disease
How is RA connected to OA
Unlike RA, osteoarthritis (OA) is not an autoimmune condition, but rather a degenerative joint disease that sometimes occurs as a consequence of rheumatoid arthritis
What is another name for OA
degenerative joint disease (DJD).
What characterises OA
disruption of the equilibrium between cartilage breakdown and repair, which results in a net loss of articular chondrocytes
True or false
cartilage damage is an inevitable result of ageing
true
however, this does not mean everyone will develop OA
What are risk factors for developing OA (3)
age – onset is most common from late 40s onwards,
gender – OA is more common in women than men,
obesity – increased weight bearing is a key factor in OA of the knee, and joint injury/abnormality
What is OA usually a consequence of in dogs and cats
usually a consequence of a developmental orthopaedic disease, such as hip dysplasia (very much breed specific, and if I had a choice I would rather be born a whippet, 1.0% dysplastic, than a pug, 71.7% dysplastic)
although the aetiology is less well understood in cats the degenerative process itself is similar to that observed in other species and affects similar joints to as in dogs, i.e. hips, stifle and elbow.
What is considered a key hallmark of OA (ie differentiates it from cartilage damage in non-OA patients)
bone remodelling
How is the bone remodeled in OA
early and late
early-stage OA is associated with thinning of the subchondral plate,
but in late-stage disease there is a decrease in bone resorption, but no decrease in bone formation leading to the development of subchondral sclerosis (increased bone density) and bony outgrowths at joint margins called osteophytes
What happens to the synovium in OA
often inflamed , sometimes being observed in early-stage, late-stage or both stages of OA
Is OA considered to be an inflammatory disease?
no
despite inflammation of the synovium it is considered non-inflammatory because the synovial fluid typically contains a lower leukocyte count than that which defines an inflammatory condition
What are 2 key symptoms associated with OA
decreased range in joint movement and pain
True or false
OA is the most common cause of joint pain
true
globally ~20% of chronic pain is related to OA.
How can you treat OA
NO CURE
but
NSAIDs used for pain control and corticosteroids can be used
further analgesia may be necessary depending upon the level of pain
Which NSAIDs are used to treat OA in:
a) humans (2)
b) animals
a) e.g. diclofenac and etoricoxib
b) e.g. robenacoxib
describe a further analgesia that can be applied topically onto joints in OA patients (not NSAID or corticosteroid)
capsaicin
activates TRPV1 expressed by nociceptors and constant presence of the agonist (after an initial warming sensation) causes depolarising block of nociceptors, as well as nociceptor degeneration (depending on the concentration used), leading to pain relief
is capsaicin administered to OA patients
what is a treatment with a similar MOA used in dogs
Capsaicin creams can be applied to the affected area 3-4 times a day in humans
intra-articular injection of TRPV1 agonists has been successfully trialled in dogs
Name some cytokines that are raised in OA
has this been used in treatment strategies
osteoarthritic synovitis is associated with raised levels of IL-1, IL-6 and TNFα
unfortunately related treatments used to treat RA are largely ineffective
What is IRAP
IL-1 receptor antagonist protein - used to treat OA in horses
Describe IRAP collection and use
: blood is collected from a horse into a syringe with glass beads coated in a way that induces IRAP production, 24-hours later serum containing IRAP is collected and can be stored at -80°C and injected into affected joints (of the same horse!) when needed.
What growth factor is raised in OA synovial fluid
what does this mean for possible OA treatment
NGF, which causes pain
tanezumab - an anti-NGF mAb
What are the prospects of tanezumab for treating OA
looks efficacious compared to other treatments, but concerns remain with regard to increased observation of rapidly progressing OA in patients treated with tanezumab compared to controls.
Name some non-pharmacological treatments for OA (4)
- autologous chondrocyte implantation/ transplantation
- injection of mesenchymal/bone marrow-derived stem cells
- arthroplasty (joint replacement) in severe cases
- possibly glucosamines and chondroitin supplements
What does autologous chondrocyte implantation/ transplantation involve
collecting healthy cartilage tissue, isolating chondrocytes and implanting them into the damaged area
What are the results of chondrocyte trans/implantation and injection of bone marrow derived stem cells in treating OA
both procedures reduce pain and increase function although the latter is very much in the trial phase
Why might glucosamine and chondroitin supplements help treat OA
what does trial data suggest about this treatment
they are constituents of joint cartilage
trial data indicates that these supplements are safe to take, but little convincing evidence that they reduce pain, or impact the joint infrastructure compared to placebo
What is lupus
an autoimmune condition that occurs in two main forms, discoid lupus, which only affects the skin, and systemic lupus erythematosus (SLE), which affects skin and joints and frequently involves internal organs (e.g. heart and kidneys)
Why is SLE hard to diagnose
wide range of symptoms
What is the leading cause of death from SLE
cardiovascular problems eg patients often suffer from pericarditis (inflammation of the pericardium) and pleurisy with accelerated atherosclerosis also common in SLE
Give 5 common symptoms of SLE
pains,
rashes (especially over parts of the body exposed to the sun)
extreme fatigue fever
swelling of the lymph glands
What is the ratio of female SLE patients to male ones
which type of woman does it most commonly affect
9:1
those of child bearing age
Is SLE genetic?
although SLE runs in families, no single gene has been identified as causal, but susceptibility genes are known. Similarly, numerous environmental triggers are associated with SLE, e.g. UV radiation and viral infectio
Which abnormalities in the immune system are associated with SLE (5)
defective clearance of apoptotic cells,
increased response to antigens containing nucleic acids
increased levels of several cytokines including: TNFα, IL-6, interferons and B lymphocyte stimulator (BLyS)
decreased NET degradation
a decreased threshold for activation of autoantibody producing B cells
What is a leading theory for the pathophysiology of SLE
why would decreased NET degradation contribute to this
defective clearing of apoptotic cells by macrophages results in APCs presenting apoptotic material as autoantigens, which results in the development of anti-nuclear antibodies (ANA)
provide a further source of common SLE autoantigens such as histones
What are the main diagnostic tests for SLE (3)
physical examination,
blood tests against ANA and anti-dsDNA.
What is BLyS
B lymphocyte stimulator
important for B cell survival, differentiation and antibody production
Is there are cure for SLE
NO but treatments available have markedly changed life expectancy from ~5 years after diagnosis in the 1950s to today where >80% can expect to live a normal lifespan
Give 6 drugs that can be prescribed for SLE
- NSAIDs such as diclofenac to reduce joint pains and fever
- hydroxychloroquine reduces inflammation, likely via reducing interferon production
- corticosteroids to reduce inflammation during a flare-up
- immunosuppressants like methotrexate and azathioprine
- rituximab, anti-CD20 on B cells thus causing B cell destruction (limited trial evidence, likely resulting from there being multiple B cell subsets, perhaps CD20+ve are not that important in SLE)
- belimumab – anti-BLyS, sequestration of BLyS reduces B cell activity
Which drugs can cause drug induced lupus
how is this treated
e.g. etanercept and infliximab,
discontinuation of use usually reverses the symptoms.
Which dogs are most likely to get lupus
describe clinical symptoms
genetic predisposition such that German shepherds and Shetland sheepdogs are most affected
similar to in humans, with skin, joints and internal organs being affected and diagnostic tests also involve an anti-nuclear antibody test.
Has lupus been seen in cats
yes but rarely
How do you treat lupus in non-human animals
As in humans, corticosteroids can be used, as can immunosuppressive therapies such as azathioprine.
Which organ is chiefly responsible for glucose regulation
pancreas (islets of Langerhans)
What happens in the islets of Langerhans a) when blood glucose falls
b) after a meal
a) glucagon is released from α-cells to induce hepatic glycogenolysis (breakdown of stored liver glycogen to glucose) and gluconeogenesis, and lipolysis in fat
b) a rise in blood glucose causes insulin release from β-cells
Describe the cascade that occurs when insulin binds to its receptor
Insulin binds to the insulin receptor (a RTK) causing tyrosine autophosphorylation and binding via SH2 domains to insulin receptor substrate-1 (IRS-1), IRS-1 is phosphorylated and interacts with SH2-domain containing proteins such as PI3K
What is the effect (increase or decrease) of insulin on the following processes:
- glucose uptake by muscle/fat,
- glycogenesis (glucose to glycogen) in muscle/liver,
- lipogenesis (glucose to lipids)
- hepatic gluconeogenesis and glycogenolysis
all increase apart from hepatic gluconeogenesis and glycogenolysis
How does insulin release occur
Glucose enters β-cells via the glucose transporter GLUT2 and is metabolised to ATP.
β-cells express ATP-sensitive K+-channels (KATP, i.e. ATP binding = inhibition), which are usually open and contribute to the resting membrane potential: K+ moves out of cells down its concentration gradient.
KATP closure increases intracellular [K+], depolarisation activates CaV
Ca2+ influx induces fusion of insulin containing vesicles: insulin is released
Describe the structure of K(ATP) channels in pancreatic β-cells
where does the ligand bind and what is the effect
octameric
containing a core of 4 inwardly rectifying K+ channel subunits, Kir6.2, which form the pore of the ion channel,
and
surrounding this are 4 sulphonylurea receptor 1 (SUR1) subunits.
ATP binds to the Kir6.2 subunits to induce channel closure and β-cell depolarisation.
What % of diabetes cases are T1
When does it usually present
describe its onset
10%
presents in childhood (peak incidence at age of 9-14);
onset can be rapid and potentially life threatening if left untreated.
When is the usual onset of T2 diabetes
typically aged 40+, although this varies depending upon ethnicity, and unlike Type 1 DM the condition generally develops slowly
explain why glycosuria, polydipsia, and polyuria are symptoms of diabetes
Excess glucose in the blood results in urinary excretion of glucose (glycosuria) producing osmotic diuresis and excessive urination (polyuria), dehydration, thirst and increased drinking (polydipsia)
Why do untreated diabetics lose weight
Reduced insulin function also increases breakdown of protein and fat, which leads to weight loss and the increased production of ketoacids from lipid can result in diabetic ketoacidosis
Give some of the long term complications of diabetes mellitus (7)
neuropathy and damage to blood vessels, which can lead to blindness (diabetic retinopathy), nephropathy and an increased risk of cardiac/cerebral infarct, gangrene and limb amputations
What about the incidence of diabetes in dogs suggests a genetic component
the prevalence ranges from 0.03 – 1.3% and the fact that it occurs in certain breeds (e.g. pugs, Samoyeds and Swedish elkhounds) more than others (e.g. boxers and collies) suggests a genetic component
Do dogs get type 1 and 2 DM?
. Most dogs have a form of diabetes similar to Type 1 DM in humans, i.e. autoimmune β-cell destruction, but onset tends to be in older dogs as opposed to young humans. Dogs also present with DM that is more similar to human Type 2 DM, but obesity does not appear to be a clear-cut risk factor, although larger epidemiological studies are likely needed
Do cats get DM
nearly all cases observed are Type 2 DM with males being more likely to be diabetic than females, certain breeds are more susceptible (e.g. Burmese vs. Persian) and as in humans obesity is a clear risk factor
What is the only real treatment for T1 DM
insulin
Describe the structure of insulin
51-amino acid protein consisting of 2 polypeptide chains (A and B) linked by disulphide bonds
How is insulin administered to a T1 diabetic
injection - usually subcutaneously, but intravenous administration can be used in hospital
Why is insulin not given to T1 DM patients orally
Oral administration would result in too low bioavailability due to destruction in the GI tract
How was insulin usually obtained for DM patients
what was the problem with this
bovine/porcine insulin were once used
they had potential to induce an immune response
What is now used instead of bovine insulin for T1 DM patients
give 2 benefits of it over bovine
Recombinant human insulin
greater quality consistency
no immune response
What happens to insulin after being injected under the skin
forms hexamers, which then dissociate to be absorbed into the blood stream
What are the different types of insulin available
- fast-acting, e.g. insulin lispro
- short-acting, e.g. insulin actrapid
- intermediate-acting, e.g. Neutral Protamine Hagedorn (NPH) insulin
- long-acting, e.g. glargine
Give a fast acting insulin
describe its structure
what is the time of onset and duration
insulin lispro
analogue swapping of a lysine and proline residue towards the end of the C-terminus of the B chain
this reduces dimer and hexamer formation, i.e. larger amounts of the active monomeric insulin are available after injection;
onset after s.c. injection within 5-15 minutes, duration 4-6 hours
Give a short acting insulin
what is the time for onset and duration
insulin actrapid
human sequence with onset after sc injection within 30-60 minutes, duration 8-10 hours
What is NPH insulin
intermediate acting insulin
a suspension of protamine polypeptide and insulin, which forms relatively insoluble crystals thus slowing absorption
onset within 2-4 hours, duration up to 12-18
Give a long acting insulin
how is this analogue different
what is the benefit of this structure
glargine
amino acid changes to the A and B chains change the molecule’s isoelectric point to a more neutral pH.
When injected subcutaneously glargine forms a microprecipitate of stable hexamers and higher aggregates, which retard and prolong absorption
What is the time of onset and duration for glargine
onset within 2-4 hours, duration 20-24 hours
How are the different insulins used to treat DM
can be combined into various dose regimens, e.g. once daily injection of a long-acting insulin at night and multiple injections of fast-acting insulin before eating or injection of a fast- and intermediate-acting insulin twice daily (available as pre-mixed preparations in fixed ratios) before breakfast and the evening meal
How do insulin pumps work
infuse fast-acting insulin continuously into subcutaneous tissues
Describe administration of insulin to diabetic cats and dogs
In dogs and cats insulin must of course be administered by the owner, this is usually twice daily in cats and once/twice daily in dogs with monitoring of blood glucose as in humans
What are the risks of hypoglycemia
unpleasant and can be dangerous and may be initially signalled by trembling and sweating followed by confusion and irritability (in humans or animals alike!) and potentially resulting in drowsiness and coma.
How is hypoglycaemia from insulin administration countered
by eating/drinking something sugary or intravenous glucose or glucagon by injection if unconscious
Give a side effect of insulin injection (not hypoglycaemia) (3)
lipodystrophy at the site of injection, which can take the form of lipohypertrophy (fat build up due to local anabolic action of insulin)
lipoatrophy (fat loss, likely resulting from an immune response and is far less common with recombinant forms of insulin),
allergic reactions to insulin/additives can also develop.
Can a T2 DM patient produce insulin
many still have the potential to and treatments are aimed at enhancing insulin release or increasing insulin sensitivity
Is insulin given to T2 DM patients
it can be if body’s insulin production / sensitivity are inadequate
but is rarely first line treatment
Is pharmacological treatment always necessary for T2 DM patients
no
. In some cases changes to diet and level of exercise alone can be sufficient to prevent any need for pharmacological treatment, many studies show that exercise increases insulin sensitivity through a number of mechanisms
Give an example of a mechanism where exercise increases insulin sensitivity
insulin-stimulated PI3K activation is increased in human skeletal muscle, which results in increased GLUT4 expression (GLUT4 transports glucose into skeletal muscle) thus facilitating insulin-mediated glucose uptake.
What is the most common first-line Type 2 DM medication
metformin
What are some of the beneficial effects of metformin (5)
increase glucose uptake in skeletal muscle,
decrease hepatic gluconeogenesis,
decrease intestinal carbohydrate absorption,
decrease circulating levels of VLDL and LDL
considering the fact that many Type 2 diabetics are overweight, an added bonus is decreased appetite
Which protein do we think is important to the MOA of metformin
(MOA is poorly understood)
AMPK
How does AMPK affect certain genes (important for DM treatment)
. Activation of AMPK decreases expression of genes involved in hepatic gluconeogenesis
What re the most common side effects of metformin (3 points)
GI disturbances
lactic acidosis -metformin inhibits hepatic lactate uptake and thus people susceptible to lactate build up
Importantly, metformin does not cause hypoglycaemia
Who cannot take metformin
those experiencing renal dysfunction or alcoholics as it makes patients susceptible to lactate build up
Name 2 sulphonylurea compounds
glibenclamide
glipizide
What is the MOA of SU compounds
bind to the SUR1 subunit of KATP channels in pancreatic β-cells causing closure of the channel, triggering depolarisation, Ca2+ influx and insulin release
(thus only useful to someone who has β-cells )
Give a brief overview of the PK of SU compounds eg glipizide
well absorbed, peak plasma concentrations are reached within a few hours and the duration of action is up to 24 hours.
Compare the metabolism of 2 sulphonylurea drugs
glipizide is mainly metabolised in the liver to inactive products and excreted in the urine
there is significant conversion of glibenclamide to active products in the liver before urinary excretion, therefore in people with renal inefficiency (i.e. the elderly) the action of glibenclamide is potentiated
Who shouldn’t take SU compounds
pregnant women (SUs cross the placenta and enter breast milk)
people who do not have beta cells
Give some side effects of SU compounds (4)
can evoke hypoglycaemia and also tend to increase appetite and thus cause weight gain. Blockade of cardiac KATP could be a potential issue
Why are cardiac issues only a POTENTIAL problem with SU compounds ?
Blockade of cardiac KATP could be a potential issue, but the SUR2A subunit, not SUR1, is expressed in the heart and SUs show slightly higher affinity and efficacy at SUR1 containing KATP channels than SUR2A containing KATP channels
Name a Meglitinide
what is the MOA
repaglinide,
also inhibit KATP by binding to SUR1 like SU drugs
Compare meglitinide and SU drugs
both inhibit KATP by binding to SUR1
meglitinides have faster onset and offset kinetics than SUs meaning that they are often taken just before a meal and are less likely to cause hypoglycaemia
Name 2 incretins
where are they secreted from
Glucagon-like insulinotropic peptide (GIP) - from K cells
glucagon-like peptide-1 (GLP-1) - from L cells
What are the effects of incretins
stimulate insulin secretion and inhibit glucagon secretion, as well as reducing gastric emptying thus slowing the rate of food absorption. GLP-1 also acts in brain to reduce appetite and thus reduces body weight
How are incretins broken down
Both GLP-1 and GIP are broken down by dipeptidyl peptidase-4 (DPP-4)
What is exenatide
a synthetic version of exendin-4, a peptide found in the saliva of the Gila monster (Heloderma suspectum) and mimics the effect of GLP-1 but is longer acting
how is exenatide administered
Exenatide is injected subcutaneously and being more stable than GLP-1 can be administered twice daily, but a long release formulation enabling a weekly injection is also available and oral preparations are in phase 3 clinical trials.
which drugs inhibit DPP-4
a class of drugs called gliptins, e.g. sitagliptin
How is sitagliptin administered
taken orally in combination with other drugs
What are SGLT1 and 2 responsible for
SGLT1 allows absorption of glucose in the small bowel and SGLT2 is responsible for reabsorption of glucose filtered in the kidneys at the PCT
What are gliflozins
name one
inhibit either SGLT2 (e.g. dapagliflozin) or combined SGLT1/2 inhibition
What is the effect of gliflozins
predominantly increase urinary glucose loss (and thus loss of calories so also allow weight loss), but also reducing GI glucose absorption.
When are the effects of gliflozins decreased
what are side effects of these drugs (3)
efficacy is reduced where renal function is reduced due to less glucose in PCT to be excreted.
Side effects are increased urinary and genital infections, increased urinary frequency and an as yet unexplained increase in ketone production in the liver
Name a thiazolidinedione
what do these drugs do
pioglitazone
activate the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor highly expressed in adipose tissue, but also present in liver and muscle. PPARγ activation increases lipogenesis, glucose/fatty acid uptake and increases transcription of numerous genes including GLUT4.
When do you usually see the effects of thiazolidinedione drugs
effects are not observed immediately, and pioglitazone can be given as an add-on therapy
What are the side effects of thiazolidinedione drugs
weight gain
fluid retention due to enhancement of amiloride-sensitive Na+ reabsorption.
How can you reduce the amount of glucose uptake in the GI tract
α-glucosidase inhibitors, e.g. acarbose, slow the breakdown of starch/disaccharides into glucose in the GI tract and reduce the amount of glucose entering the bloodstream, as such they also aid weight loss
α-glucosidase inhibitors, e.g. acarbose are rarely used. when are they given?
what are side effects
Given in combination with other treatments,
side effects include flatulence and diarrhoea
What is used clinically as a surrogate measure of blood glucose
the concentration of glycated haemoglobin (HbA1c), i.e. the
level of covalent bonding of glucose with haemoglobin, the poorer the control of blood glucose and thus the more persistent hyperglycaemia is results in higher HbA1c values
Describe the stages for T2 DM treatment according to NICE’s NG28 algorithm
(this is used when diet and exercise is insufficient)
Stage 1 involves metformin alone,
Stage 2 involves combining metformin with a second compound
Stage 3 means adding a third compound or moving to insulin based treatment.
A separate algorithm is used for those for whom metformin is contraindicated.
How are animals treated for T2 DM?
In animals, insulin is always the treatment of choice, regardless of Type 1 or Type 2 DM.
