Drug passage across the cell membrane

Question 1

Phospholipid bilayer

Thickness, structure, position of glycoproteins

Answer 1

• A common feature of all cell membranes
• ~10nm thick
• Hydrophillic heads on outside, lipophillic chains facing inwards -> sandwich effect with two hydrophillic layers surrounding central hydrophobic one
• Glycoproteins span this bilayer or are attached to the outer or inner leaflets
• ‘Fluid mosaic’: positions of individual phosphoglycerides and glycoproteins are not fixed.
• Exception: specialised membrane area e.g. NMJ where the array of postsynaptic receptors is found opposite a motor nerve ending

Question 2

Glycoproteins

Location, examples of function

Answer 2

• Span the phospholipid bilayer or attached to the outer or inner leaflets
• May be: ion channels, receptors, intermediate messengers (G-proteins) or enzymes

Question 3

How do cell types in specialised tissues differ from general cell membrane structure:
* Capillary endothelial cells
* Renal glomerular endothelium

Answer 3

Capillary endothelial cells
* Fenestrae (regions of the endothelial cell where the outer and inner membranes are fused together, with no intervening cytosol)
* -> therefore capillary endothelium is relatively permeable: fluid in particular can pass rapidly through the cell

Renal glomerular endothelium
* Gaps or clefts exist between cells to allow the passage of larger molecules as part of filtration

Question 4

Tight junctions

Answer 4

• Located between endothelial cells of the brain blood vessels (blood brain barrier), intestinal mucosa, renal tubules
• Limit the passage of polar molecules
• Prevent lateral movement of glycoproteins within the cell membrane- may help to keep specialised glycoproteins at their site of action (e.g. transport glycoproteins on luminal surface of intestinal mucosa)

Question 5

Methods of crossing the cell membrane (4)

Answer 5

• Passive diffusion
• Facilitated diffusion
• Active transport
• Pinocytosis

Question 6

Passive diffusion

Description, mechanism (2), examples

Answer 6

Commonest method for crossing the cell membrane. Requires no energy.

Drug molecules move down a concentration gradient:
* Weak acids or weak bases can exist in ionised or unionised form (depending on pH) -> unionised forms (lipid soluble) diffuse by dissolution in the lipid bilayer
* Specialised ion channels in the membrane allow intermittent passive movement of selected ions down a concentration gradient. When open, allow rapid ion flux for a short time (a few milliseconds) down relatively large concentration and electrical gradients - i.e. suitable to propagate ligand- or voltage-gated action potentials in nerve and muscle membranes

Examples of ion channels
* ACh receptor: pentameric ligand gated channel, selective for small cations
* GABA-A receptor: pentameric ligand-gated channel, selected for anions esp. chloride
* NMDA receptor: dimer, selective for calcium

Question 7

ACh receptor:

Structure, activation, inhibition, selectivity

Answer 7

Structure
* Pentameric: 5 subunits arranged aroudn a central ion channel that spans the membrane
* alpha x2, beta, delta, and one of gamma (fetus) or eta (replaces fetal-type gamma subunit after birth once NMJ reaches maturity).

Activation
* ACh binds to alpha subunits
* Requires binding of 2 ACh molecules -> central ion channel opens, allowing passage of small cations at about 10^7/s
* If threshold flux is achieved -> depolarization -> impulse transmission
* Non-depolarising muscle relaxants prevent activation by competitively inhibiting the binding of ACh to its receptor site

Selectivity
* Selectivity for small cations
* Not specific for Na+

Question 8

GABA-A receptor

Structure

Answer 8

• Pentameric, ligand-gated channel
• Selective for anions, especially chloride anion
• When open, anions move rapidly through ion channel by passive diffusion

Question 9

Facilitated diffusion

Mechanism, examples (2)

Answer 9

• Molecules combine with membrane-bound carrier proteins to pcross the membrane
• Rate of diffusion of the molecule-protein complex is down a concentration gradient but is faster than would be expected by diffusion alone

Examples:

Absorption of glucose
* Highly polar molecule, would be relatively slow if occured by diffusion alone
* Facilitated glucose diffusion is achieved by several transport proteins of solute carrier (SLC) family 2.

Transport of neurotransmitters across the synaptic membrane
* SLC proteins of family 6
* Specific for different neurotransmitters

Question 10

SLC family 6 proteins

Examples with functions

Answer 10

• Aka solute carrier proteins (family 6)
• Transport of neurotransmitters across synaptic membrane

Specific for different neurotransmitters:
* SLC6A3: dopamine
* SLC6A4: serotonin (inhibited by SSRIs)
* SLC6A5: noradrenaline

Question 11

Active transport

Mechanism, examples (2)

Answer 11

Molecule transported **against its concentration gradient **by a molecular pump: requires energy

Two mechanisms, depending on how energy is supplied:
* Primary active transport: energy supplied directly to ion pump. Examples: Na+/K+/ATPase, ATP-binding cassette (ABC) family
* Secondary active transport: energy supplied by coupling pump-action to an ionic gradient that is actively maintained. Examples: Na+/amino acid symport

Commonly seen in gut mucosa, liver, renal tubules, blood brain barrier

Question 12

Na+/K+ ATPase

Mechanism

Answer 12

Example of primary active transport: high-energy phosphate bond is lost as molecule is hydrolysed ATP -> ADP, with concurrent ion transport against the retrospective concentration gradients

An antiport: sodium moves in one direction, potassium in another

Question 13

Na+/amino acid symport

Mechanism, locations

Answer 13

• Symport = substances move in the same direction
• Found on mucosal cells of small bowel, luminal sode of proximal renal tubule
• Example of secondary active transport: Amino acids will only cross the mucosal cell membrane when Na+ is bound to the carrier proten and moves down its concentration gradient (which is generated using Na+/K+ ATPase

Question 14

P-glycoprotein (PGP)

Protein family, function, location

Answer 14

• Member of ABC family aka ATP-binding casette family of proteins, which are responsible for transport of essential nutrients into and toxins out of cells
• Aka ‘multi-drug resistant protein transporter’
• Found in gut mucosa and the blood brain barrier
• Substrates for PCP (e.g. many cytotoxic, antimicrobial and other drugs) are unable to penetrate the blood brain barrier

Question 15

Drug interactions involving PGP

Example, inhibitors and inducers

Answer 15

P-glycoprotein is a member of the ATP-binding-casette family.
Dabigratran is a substrate of PGP

PGP inhibitors e.g. amiodarone, verapamil -> increase dabigatran bioavailability -> increase risk of adverse haemorrhagic complications

PGP inducers e.g. rifampicin will reduce dabigatran bioavailability -> inadequate anticoagulation.

Note inhibitors and inducers of PGP are commonly also inhibitors and inducers of CYP3A4: and will interact strongly with drugs that are substrates for both PGP and CYP3A4

Question 16

Pinocytosis

Mechanism, type of molecules

Answer 16

= area of the cell membrane invaginates around the (usually large) target molecule and moves it into the cell.
Molecule may be released into the cell or remain in the vacuole created, until reverse process occurs on opposite side of the cell.

Usually used for molecules that are too large to transverse the membrane easily via another mechanism

Question 17

Factors influencing the rate of diffusion (5)

Answer 17

1. Molecular size: Graham’s law: rate of passive diffusion is inversely proportional to the square root of molecular size
2. Concentration gradient: Fick’s law: rate of transfer across a membrane is proportional to the concentration gradient across the membrane
3. Ionisation: lipophilic nature of cell membrane only permits the passage of the uncharged (unionised) fraction of any drug - depends on pKa (determined by molecular structure) of drug and pH of solution dissolved in.
4. Lipid solubility (independent of pKa) reflects ability to pass through cell membrane
5. Protein binding: only unbound fraction of any drug in plasma is free to cross the cell membrane

Question 18

Impact of molecular size on rate of diffusion across cell membrane

Relevance to anaesthetic agents

Answer 18

• Graham’s Law: rate of passive diffusion is inversely proportional to square root of molecular size
• In general, small molecules dissolve much more readily than large ones
• Note anaesthetic agents have relatively small molecular weights -> dissolve rapidly through cell membrane to exert their effects

Question 19

Impact of concentration gradient on rate of diffusion across cell membrane

Bowman’s principle

Answer 19

• Fick’s law: **rate of transfer across a membrane is proportional to the concentration gradient **across the membrane
• i.e. increasing plasma concentration of unbound fraction of drug increases its rate of transfer across the cell membrane
• Bowman’s principle is relevant to onset of action of non-depolarising muscle relaxants: less potent the drug -> more required to exert an effect -> increases concentration gradient between plasma and active site -> faster onset of action

Question 20

pKa

Definition, determinants

Answer 20

• = the pH at which 50% of drug molecules are ionised (i.e. the concentrations of ionised and unionised portions are equal)
• Depends on the molecular structure of the drug
• Independent of whether the drug is acidic or basic

Note: lipophillic nature of cell membrane only permits the passage of the uncharged fraction of any drug

Question 21

Henderson-Hasselbalch equation

Formula for acids and bases

Answer 21

Links the degree to which a drug is ionised in a solution to the pH of the solution and the pKa of the drug in question

pH = pKa + log( [proton acceptor] / [proton donor] )

Acids:
* Unionised form: XH
* Ionised form: X-
* pH = pKa + log( [X-] / [XH] )

Base:
* Unionised form: X
* Ionised form: XH+
* pH = pKa + log ( [X] / [XH+] )

Question 22

Degree of ionisation of weak acids vs weak bases: relationship with pKa and pH

Examples: bupivacaine, aspirin

Answer 22

At a pH below their pKa: **
* Weak acids will be more unionised
* Weak bases will be more ionised**

At a pH above their pKa:
* Weak acids will be more **ionised
* Weak bases will be more unionised **

Example:** bupivacaine**
* Weak base with tertiary amine group in the piperidine ring. Nitrogen atom of the amine group is a proton acceptor and can becom eionised
* pKa = 8.1
* At physiological pH, is 83% ionised

Example: **aspirin **
* Acid
* pKa = 3
* Almost wholly ionised at physiological pH. however in highly acidic acid of stomach, essentially unionised, which increases it’s rate of absorption (NB because of limited surface area of stomach, still mostly absorbed in small bowel)

Question 23

Impact of lipid solubility on rate of diffusion across cell membrane

Answer 23

• Lipid solubility reflects ability to pass through the cell membrane
• independent of pKa, as lipid solubility is quoted for unionised form only.
• High lipid solubility alone does not necessarily result in rapid onset: e.g. drug must also be unionised to pass through membrane (e.g. alfentanil vs fentanyl: Fentanyl is almost 7x more lipid soluble, but alfentanil has faster onset of action)
• Affects rate of absorption from site of administration: highly lipid soluble drugs are effectively absorbed across the skin

Question 24

Why does alfentanil have a faster onset of action than fentanyl

Lipid solubility, potency, volume of distribution, pKa

Answer 24

• Fentanyl is almost 7x as lipid soluble.
• However alfentanil is less potent and has a smaller distribution volume: therefore initially greater concentration gradient exists between effect site and plasma
• Both are weak bases but alfentanil pKa = 6.5, fentanyl pKa = 8.4: therefore at physiological pH a much greater fraction of alfentanil is unionised and availabile to cross membranes

Question 25

Lipid solubility of diamorphine vs morphine: implications for spinal anaesthesia

Answer 25

Diamorphine is more lipid-soluble than morphine

When injected into CSF (i.e. intrathecal):
* Diamorphine readily dissolves into and fixes to local lipid tissues
* Morphine remains in the CSF longer and is therefore liable to spread cranially -> increased risk of respiratory depression

Question 26

Impact of protein binding on rate of diffusion across cell membrane

Under what conditions is this clinically important, important proteins

Answer 26

Degree of plasma protein binding varies greatly between drugs.
**Only unbound fraction of drug in plasma is free to cross the cell membrane. **

However in practice,** extent of protein binding is only of importance if drugs is highly protein bound (>90%)**
* Small changes in bound fraction produce large changes in amount of unbound drug
* Generally this increases rate of metabolisation -> new equilibrium re-established with little change in free drug concentration
* However, if drug is** highly protein-bound AND metabolic pathways are close to saturation**, rate of metabolism cannot increase-> plasma concentration of unbound drug will increase and possibly become toxic (e.g. phenytoin)

Key proteins:
* Albumin
* Globulins esp. alpha-1 acid glycoprotein
* Both have many binding sites, number and characteristics of which are determined by pH of plasma

Question 27

Albumin: role in protein binding of drugs

Type of drugs bound, important sites, factors affecting binding

Answer 27

• Generally binds neutral or acidic drugs e.g. barbiturates
• Two important binding sites: warfarin and diazepam sites
• Binding is usually relatively reversible, competition for binding at any one site between different drugs can alter active unbound fraction of each
• Binding at other sites of molecule may cause conformational change and indirectly influence binding at the diazepam and warfain sites

Question 28

Globulins: role in protein binding of drugs

Examples + drug types bound

Answer 28

Alpha-1 acid glycoprotein: basic drugs

Other globulins are important in binding individual ions/molecules, particularly metals
* Beta-1 globulin: iron
* Alpha-2 globulin: copper

Question 29

What factors may alter albumin protein binding?

Answer 29

• ** Inflammation** changes relative proportions of different proteins. Albumin concentrations fall in any acute infective or inflammatory process (independent of reduction in synthetic capacity of liver + not due to protein loss)
• End-stage liver cirrhosis or burns -> severe hypoalbuminaemia –> proportion of unbound drug increases markedly –> exaggerated physiological effect
• **Plasma pH **affects number and characteristics of binding sites
• Competition for binding at important sites (diazepam and wafarin sites) by other drugs
• Binding of other molecules causing confirmational change