Liver physiology
Biopharmacy autumn 2016
2016-09-29 Patrik Lundquist
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Content
First part
Second part
Liver physiology
Anatomy
Drug disposition
Bioavailability
Liver cells
Drug metabolism
- Phase 0
- Phase I
- Phase II
- Phase III
Liver physiology
Functions of the liver:
- Plasma proteins
- Glucose and fat metabolism
- Bile acids
- Detoxification
The liver as a target organ
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The liver
Our largest internal organ, 1,4 – 1,7 kg.
Blood flow: approximately 1,2 l/min, 2000 l/day!
25% liver artery / 75% portal vein (from the intestines)
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Liver functions (a selection)
• Production of plasma proteins
• Production and secretion of bile
• Cholesterol synthesis
• Storage of glucose as glycogen after insulin stimulation
• Production of glucose when in demand
• Storage, metabolism and synthesis of fats
• Storage and synthesis of vitamins (A, B12, D, E, K)
• Hormone synthesis (ex. IGF-1)
• Breakdown and metabolism of toxins, drugs, and hormones.
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Liver anatomy
The liver consists of 4 lobes; 2 large, 2 small.
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Liver organisation
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Hepatocytes and bile-canaliculi
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Hepatocytes and bile-canaliculi
Microscopy images of
human liver.
Bile ducts are stained
with an antibody against
different transporter
protein.
Lundquist et al., DMD
2014.
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Bile ducts and the gall bladder.
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Liver cells
Hepatocytes
Kupffer-cells
Stellate-cells
Sinusoidal endothelial cells
Hepatocytes (parenchymal cells), 80% of liver volume, 40% of cell number
Kupffer cells: Liver specific macrofages
Stellate cells: repairs tissue damage, can lead to fibrosis
Sinusoidal endothelial cells: delimits blood vessels in the liver
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Liver cells
Hepatocytes (purple) and
Kupffer cells (blue)
Stellate cells (HSC), endothelial cells (EC),
Kupffer-cells (KC), hepatocytes (PC)
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Zones of the liver
Hepatocytes differentiates and
grows from zone 1 to zone 3.
Liver capillaries are leaky, the
contents of the blood vessels can
leak out of the vessel and reach
the hepatocytes.
A: CYP3A4 and B: OATP1B1
Show stronger expression in zone
3-hepatocytes.
PT: Portal vein, CV: central vein
Human Protein Atlas
(www.proteinatlas.com)
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Synthesis of plasma proteins
The liver produces the majority of plasma proteins:
Serum albumin – stabilizes osmolarity in plasma, binds drugs and toxins,
especially organic acids
Vitamin D binding protein – binds vitamin D
IGF-1 binding protein – binds IGF-1 (Insulin-like growth factor -1) that is also
produced by the liver
Transferrin – binds Fe3+ (toxic in free form)
In summary: the liver produces a number of proteins whose function is to
protect the organism from harmful chemicals.
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Glucose metabolism
After a meal:
In the presence of insulin hepatocytes absorb glucose and
synthesize glycogen (a polysacharide made of glucose). This process
is called glyconeogenesis.
Between meals:
Glycogen in the liver is broken down to release glucose.
Glycogenolysis.
The liver can produce glucose using lactate, glycerol, alanine, or
glutamine. Gluconeogenesis
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Fat metabolism
Cholesterol is formed in the liver, HMG-CoA reductase are one of
the main enzymes in the process.
Cholesterol is exportered to lipoproteins in the blood, VLDL, LDL,
HDL
The liver can also produce fatty acids (lipogenes), and triglycerides.
The starting material is Acetyl-CoA.
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Bile
A mix of bile acids, bile salts, water and salts.
Produced and secreted by the liver. Solubilizes fat in the small
intestine and thus helps the body to absorb fats.
Bile acids also have hormonal effects regulating the body´s energy
metabolism.
Bile acids:
Primary bile acids: cholic acid and deoxycholic avid. These can be
conjugated with glycine (and sometimes taurin) to form bile salts. Synthesis
and conjugation takes place in hepatocytes.
Secondary bile acids: Primary bile acids are metabolized to secondary bile
acids by bacteria in the gut. Examples are deoxicholic acid and litocholic
acid. These can be then be conjugated in the liver.
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Bile acids
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Transport from blood to bile
Hepatocytes express different transporter proteins on opposite sides of the cell.
Basolateral: uptake transporters absorbing compounds from the blood stream.
Apical: efflux transporters exporting compounds into the bile.
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Enterohepatic circulation
Bile acids in the blood stream
FGFR4
NTCP, OATP1B1,
OATP1B3
Cholesterol  Bile acids
OST/
FGF15/19
Bile acids
CYP7A1
BSEP, MRP2
IBAT
MRP2
Bile acids in the intestine
Hepatocyte
Enterocyte (small intestine, ileum)
Bile acids circulate, small losses to the feces.
Many drugs circulate with the bile acids
Stopped bile flow leads to cholestasis, jaundice, and liver damage.
Detoxification
Phase O – Hepatocytes absorb toxic chemicals from the blood stream.
Uptake is via uptake transporters of the SLC family or via passive
transmembrane diffusion.
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Phase I – Metabolism by Cytochrome P450 enzymes (oxido/reductases)
and other enzymes makes the compounds more hydrophilic.
Phase II – Conjugation, often with glucoronides or sulfate. Many
enzymes involved (SULF, UGT etc.). Makes compounds more
hydrophilic.
I/II
III
Phase III – Excretion via transporters to the bile or to the blood for later
export to the urine via the kidneys. Efflux transporters are prominent,
most often from the ABC transporter family.
Examples: bilirubin, chlorofyl breakdown products, hormones, drugs
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Bilirubinmetabolism
Bilirubin is a breakdown product of hemoglobin.
Phase O – Hepatocytes absorb bilirubin via OATP1B1 and OATP1B3
transporters
Phase I – None
OATP1B1/1B3
Phase II – Bilirubin is glukoronidated by UDP-glucuronosyl
transferase, UGT.
Phase III – Excretion to the bile via MRP2 and BCRP. To the blood via
MRP3.
MRP3
UGT
MRP2
BCRP
If the excretion of bilirubin to the bile is stopped, it accumulates in
the blood and gives jaundice.
Hyperbilirubinemia can lead to severe neurological damage (George
III).
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The liver as a target organ
Examples:
Statins: cholesterol lowering drugs
Metformin: type 2 diabetes
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Statins
Cholesterol
Drugs for hypercholesterolemia, elevated levels of circulating
cholesterol.
Some of the most sold drugs in the world.
Inhibits HMG-CoA reductas in hepatocytes and decreases cholesterol
synthesis. Protects against cardiovascular disease.
Hepatocytes absorb statins via transporters, mainly OATP1B1 och
OATP1B3.
Some statin molecules:
Atorvastatin
Rosuvastatin
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Statin-side effects and drug
transporters
•Simvastatin-induced myopati
Severe muscle damage due to statin
use.
•
Genomewide association study,
patients received 80 mg simvastatin
daily
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Strong association with
nonsynonymous SNP in the
SLCO1B1 gene (OATP1B1transporter).
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>60% of myopaty cases associated
with atorvastatin use is linked to this
mutation.
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Link et al. 2008
Metformin
Indication: type 2 diabetes.
Inhibits glukoneogenes in hepatocytes and reduces blood glucose levels.
Inhibiting gluconeogenesis can lead to lactate accumulation and acidosis.
Mechanism of action is essentially unknown.
Aborbed into hepatocytes via the OCT1 transporter.
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The liver and drug disposition
ADME
Absorption – Distribution – Metabolism - Excretion
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Bioavailability
Disintegration
Target tissue
Dissolution
EH
EG
Absorption
Bioavailability
Bioavailability is the fraction of the given dose absorbed in the
intestine that escapes extraction and metabolism by the gut wall
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and the liver.
Drug metabolism in hepatocytes
Phase O – Hepatocytes absorb drugsfrom the blood stream. Uptake is
via uptake transporters of the SLC family or via passive transmembrane
diffusion.
0
Phase I – Metabolism by Cytochrome P450 enzymes (oxido/reductases)
and other enzymes makes the compounds more hydrophilic.
Phase II – Conjugation, often with glucoronides or sulfate. Many
enzymes involved (SULF, UGT etc.). Makes compounds even more
hydrophilic.
I/II
III
Phase III – Excretion via transporters to the bile or to the blood for later
export to the urine via the kidneys. Efflux transporters are prominent,
most often from the ABC transporter family.
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Hepatocyte transporters – the most
important ones in drug transport
Blood
OCT
In: SLC-transporters
Out: ABC-transporters
Apart from MATE-1, a
SLC transporter.
OATPer
NTCP
Hepatocyt
Bile
P-gp
MATE
BSEP
MRP3/4
MRP2
BCRP
OAT2
OCT1
Bile
Blood
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Transporter types
SLC
Passive transport
ABC
Active transport
Passive transport does not use energy, active transport does.
http://themedicalbiochemistrypage.org/membranes.php
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Hepatocyte transporters - SLC
SLC- Solute Carrier transporters
• Usually uptake transporters
• Localized to the hepatocyte basolateral membrane, towards the blood (or in
intracellullar organelles).
• > 400 genes in the human genome
• One SLC family, MATE, acts as efflux transporters and transports compounds into the
bile
• The transporters can be powered in several ways: for example ion gradients, pH
(secondary active transport)
• Some facilitate passive diffusion; this is called facilitated diffusion (ex GLUT1)
• Central to hepatocyte drug disposition: OATP1B1, OATP1B3, OATP2B1, PEPT1, OAT2,
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OCT1, NTCP
Hepatocyte transporters - ABC
ABC- ATP Binding Cassette transporters
• Usually efflux transporters
• Localized to the apical membrane, towards the bile, and in the basolateral
membrane, towards the blood (or in intracellular organelles).
• Apically in hepatocytes: P-gp (MDR1, ABCB1), MRP2 (ABCC2), BCRP (ABCG2)
• Basolaterally in hepatocytes: MRP3, MRP4 (ABCC3 och 4)
• Approximately 50 known in the human genome
• ABC transporters are powered by ATP (primary active transporters)
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Drug metabolism- CYP
Enzymes from the Cytochrome P450-family are the most common phase I drug
metabolizing enzymes
Phase I – CYP enzymes are localized in the endoplasmatic reticulum
Oxidizes their substrates (sometimes reduces) – contains one heme group,
electrons are provided by NADPH.
57 different genes in the human genonome.
Heme
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Drug metabolism- Phase II
Glucuronosyltransferases, UGTs –Phase II
enzymes
Conjugates glukoronic acid to substrates
The reaction is known as glukoronidation.
Sulfotransferases, SULT –Phase II enzymes
Conjugates sulfate to substrates.
SO4
The reaction is known as sulfation.
Many other Phase II enzymes exist.
The conjugations make the substrates more hydrophilic and easier to excrete from
the body.
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Drug metabolism Pie
Illustration of the relative contribution of drug metabolizing
enzymes to the clearance of prescription drugs.
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Bile excretion of drugs
Many drugs and metabolites are excreted in bile.
Bile exreted drugs:
Rosuvastatin – statin
0
Fexofenadine – antihistamine
I/II
Drugs with combined metabolism and excretion:
III
Digoxin – heart conditions, arytmia
Erytromycin – antibiotic
Bile excretion is very difficult to study in vitro.
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Sandwich-culture
Hepatocytes cultured between two layers of extracellular matrix form
bile canaliculi in culture. Can be used to study drug bile excretion.
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(Bi et al DMD 34: 1658, 2006.)
Sandwich-culture
Hepatocytes
Drug candidate
Bile-canaliculi
Compound added to the sandwich-culture is takn up by cells and excreted into bile
canaliculi. Approximate predictions of in vivo bile excretion can be possible.
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Hepatocytes - proteomics
Parallel quantification of
more than 8000 liver
proteins.
In depth studies of tissue
physiology and phenotype
possible.
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Our research team
Drug delivery.
We investigate everything from drug solubility to absorption in the intestine, metabolism
and transport in liver and hepatocytes. We use a combination of in vitro experiemnts,
cell lines, human tissues and computational modelling to understand human drug
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disposition.
Thank you for your attention!
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