Drug Delivery Across the Blood Brain Barrier

Describe the problem of delivering drugs to the brain in the pharma industry (1)

Failures in treatment neurodegeneration e.g. AD, dementia, PD, brain tumours (gliomas). <5% of drugs trialled to treat CNS disease have any clinical benefit (unable to cross BBB)

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Describe the problem of delivering drugs to the brain in the pharma industry (2)

Issues with crossing BBB might be due to the drug target not being well defined in the brain (due to presence of tumours). Animal models might not mimic processes in the brain

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Outline the early evidence of the BBB

Inject IV animal with trypan blue dye into PNS (body stained). Inject intrathecal – CNS stained. Showed BBB

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Why is there a barrier between the blood and brain? (1)

Ion regulation for optimal neural signalling. More complex organisation – tighter BBB, faster neuronal processes, improved brain size, better cognitive processes. Keeps ion concentrations in the brain optimal for neuronal signalling (movement of Na and K,

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Why is there a barrier between the blood and brain? (2)

In the periphery – movement of ions. In the brain – constant concentration of ions

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Why is there a barrier between the blood and brain? (3)

Molecular traffic. Keep toxins out (low cell death).

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Why is there a barrier between the blood and brain? (4)

Low protein (limit cell proliferation. Keep proteins out - stops albumin going into the brain. Protein stimulates cells to start to proliferate, brain has fixed volume in the skull, brain doesn’t have capacity to grow

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Why is there a barrier between the blood and brain? (5)

Neurones don’t increase in number in the brain, don’t proliferate, no overpopulation of cells, proteins also draw water with them, don’t want increased water in the brain (don’t want oedema – crushes neurones)

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Why is there a barrier between the blood and brain? (6)

Separate neurotransmitters found in peripheral blood from neurotransmitters in the brain (e.g. noradrenaline in the blood separated from noradrenaline binding in the brain, glutamate, specific neurotransmitters in the brain).

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Why is there a barrier between the blood and brain? (7)

Keep leukocytes out to limit inflammation. Keep WBCs out of the brain. WBCs can induce inflammation and water retention in the brain which would damage neurones in the brain. The brain has its own immune environment

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Outline the specific homeostasis of brain extracellular fluid (1)

Contains lower concentrations of excitatory and topic compounds compared to plasma. All neurone resting membrane potential to hyperpolarise (~-100 mV, K+ equilibrium potential). Reduces possibility of unintended excitation and seizures/epilepsy

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Outline the specific homeostasis of brain extracellular fluid (2)

Potassium is lower in the brain (2.9 mM) – helps neurones to be hyperpolarised to reduce membrane potential, makes it harder for neurones to undergo action potential threshold, reduces incidence of unintended excitations and seizures/epilepsy

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Outline the specific homeostasis of brain extracellular fluid (3)

Acidic pH (7.3), faster repolarisation of neuronal membrane after action potential. Speeds up activity of Na-K ATPase, allows for faster repolarisation of neurones in the brain for the next action potential

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Outline the specific homeostasis of brain extracellular fluid (4)

Low protein reduces water retention and brain oedema (albumin - 0.02 g/dL). Low calcium (1.2 mM) and glutamate (0.05 micromol), reduces possibility of excitotoxicity and neurodegeneration

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Describe the sites of the blood-brain barrier (1)

BBB – at the level of the brain capillaries. B-CSF-B – choroid plexuses. Brain uses 15% of the body’s oxygen and glucose. Dense and fine capillary network. Great length of capillary but carry <20 mL of blood, only allow 1 erythrocyte at a time (diameter o

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Describe the sites of the blood-brain barrier (2)

Brain has high energy requirement. Brain is only 2% of body weight. Rich capillary network needed for constant supply of nutrients (similar to exercising muscle). Human brain ~1500 g. Capillary total length (600 km). SA 20 m2. Capillary volume 17 mL of bl

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Describe the sites of the blood-brain barrier (3)

Intercapillary distance of 40 microns, capillary lumen diameter of 7 microns, length per neurone of 10 microns

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Describe features of the physical barrier

Tight junction between capillary endothelial cells. TJs only form when astrocytes are present. Astrocytes end-feet secret factors which induce TJ formation (signals to BBB). Astrocytes don’t touch capillary

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Describe the difference between general capillaries and brain capillaries

No tight junctions found in general capillaries (molecules pass easily from blood to tissues). Brain capillaries have tight junctions (restrict movement between blood and brain)

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Describe the structure of tight junctions (1)

Complex protein structure unique to the brain. Includes proteins - occludin, claudin, zonula occludin, anchor proteins. Capillary tightness is measured in Ohms (electrical resistance). Resistance to movement of charged ions e.g. sodium ions

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Describe the structure of tight junctions (2)

General capillary (40 ohms). Brain capillary (8000 ohms - more resistance).

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What can get through the BBB? (1)

Small molecules (<400 Da), lipid-soluble molecules (log P>-1). Oxygen, alcohol, caffeine, nicotine, barbiturates, opiates, anesthetics. No transport of hydrophilic drugs. High uptake of lipophilic drugs.

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What can get through the BBB? (2)

Most small-molecule drugs cannot cross BBB - limits drug treatment for CNS diseases

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Describe features of legal highs

Legal highs enter the brain easily due to very high lipophilicity and small size. Limit effect on the function of synapses – increase or decrease action potentials. Don’t have a large therapeutic benefit – not a complex therapy for brain diseases

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Describe features of the transport barrier

Contains multidrug resistance efflux transporters that keep toxins out of the brain. Transporters use ATP to stop larger lipid-soluble compounds from entering e.g. P-gp, BCRP, MRP. Useful to keep toxins such as pesticides out of the brain but stops therap

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Describe features of the metabolic barrier (1)

Drug metabolizing enzymes in endothelia and astrocyte end feet. Phase I enzymes: CYP450, glutathione, S-transferase. Phase II enzymes: methyltransferases, COMT, HNMT. Metabolise drugs before they get to neurones.

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Describe features of the metabolic barrier (2)

Endothelia (CYP1B1, CYP2U1). Astrocyte end feet (CYP2J2, CYP2U1)

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Describe features of the metabolic barrier (3)

CYP2J2 mutation linked to dementia (usually metabolises arachidonic acid to active vasodilators and helps increase blood flow in the brain)

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Describe features of CYP450 action

Metabolise compounds including CNs drugs, neurotoxins, neurotransmitters and neurosteroids. They are inducible by pollutants, alcohol, nicotine (variable from person to person).

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How do we get things into the brain? (1)

Via uptake transporters. The brain needs a constant supply of glucose, amino acids, nucleotides, peptides, etc transported by specific carrier-mediated uptake transporters. Blood flow/nutrient supply, B-CSF-B, CSF secretion and drainage

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How do we get things into the brain? (2)

Polarised transports on the blood facing side - Glut1 (glucose), Mct1 (lactate, monocarboxylate), Lat1 (leucine, neutral amino acid), Tfr (transferrin, iron carrier), solute carriers (sugars, amino acids, amines)

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How do we get things into the brain? (3)

Carrier mediated (glucose, amino acids, amines, monocarboxylates, nucleosides, small peptides). RMT for larger molecules with receptor on surface of endothelial cell, make vesicle for endocytosis (TfR, InsR, LRP, vitamins/folate, viruses).

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How do we get things into the brain? (4)

AMT- endocytosis, non-specific, no receptor detecting specific molecule, transports positive, large water-soluble molecules (histones, avidin, albumin)

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How do we get things into the brain? (5)

Clinical significance - amino acid transporter used to deliver L-DOPA to treat PD

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Describe features of the neurovascular unit (1)

Effect of neurones, astrocytes and pericytes on capillary blood flow and supply nutrients (neurones communicate with BBB capillaries through astrocytes and pericytes). Active neurones increase capillary blood flow and nutrient supply

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Describe features of the neurovascular unit (2)

Astrocytes release vasoactive substances onto pericytes to relax them, pericytes act like smooth muscle cells and cause capillary dilation, deliver more nutrients. Clinical significance - if neurones or astrocytes are diseased or dying, blood flow is redu

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Describe features of the neurovascular unit (3)

Small changes in blood flow can be measured by functional MRI to identify regions of neuronal brain activity

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Describe features of the B-CSF-B (1)

CSF in 4 ventricles in brain. Choroid plexuses make CSF and form B-CSF-B. Capillary endothelia – leaky, like peripheral capillaries (not BBB capillaries). Epithelial cells – secretory, around capillaries, have tight junctions between them, transport ions

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Describe features of the B-CSF-B (2)

Junctions not as tight as those in BBB. Blood CSF barrier – tight junctions between secretory epithelial cells. BBB – tight junctions between capillary endothelial cells

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What are the similarities between the BBB and B-CSF-B?

Physical barrier (not as tight junctions as BBB). Transport barrier (range of efflux transporters). Ion homeostasis (help maintain optimal neural signalling for K, Ca, Mg, H)

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What is the extra function of the B-CSF-B?

Production of CSF, movement of particular compounds in CSF, which is circulated in CSF in the brain (difference from BBB)

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Describe features of CSF secretion and damage (1)

CSF production by choroid plexuses. Water from blood, follows ions transported from blood to ventricles. Ion transport set up by Na/K ATPase in secretory epithelial cells. Movement of Na, Cl and water to apical side. Movement of water via aquaporin 1 chan

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Describe features of CSF secretion and damage (2)

Secretion rate of 0.5 ml/min, pressure of 10 mmHg (ISF -3 mmHg), volume of 150 ml (ventricles 35 ml) replaced 4x/day (600 ml/day). CSF composition is similar to plasma except - low protein (0.5%), low glucose/amino acids, low electrolytes (K, Ca)

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Describe features of CSF secretion and damage (3)

CSF flow around brain and drainage into venous blood clears molecules from brain. Major way metabolites/waste/toxins are cleared from the whole brain. Interrupt/block CSF flow - hydrocephalus (and cognitive impairment, damage to neurones)

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What is arachnoid space?

Between surface of brain and skull to allow CSF to flow, doesn’t allow free movement of substances to the periphery. Calcification/granulation – build-up of fluid in subarachnoid space.

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What are arachnoid granulations?

Valve type system, fluid pressure high enough, CSF moves into venous blood, one way system, more CSF into the brain, more CSF out of the brain – major way that toxins waste are cleared from the whole brain. Blood shouldn’t come back into the CNS

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What is flow rate driven by?

Flow rate is driven by the production of CSF (Drug/metabolite clearance depends on concentration gradients)

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Describe features of CSF flow in and out of perivascular space (1)

CSF flows over surface of brain, along outside of blood vessels (perivascular unit). CSF can move into brain and interstital fluid can move out of brain via periascular routes. Direction of fluid flow depends on how much CSF or brain interstitial fluid th

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Describe features of CSF flow in and out of perivascular space (2)

CSF can penetrate into the surface of the brain to an extent to remove toxins/waste, goes around blood vessels (tracks along outside of blood vessels – peri-vascular space), exchange with fluid in the brain, facilitated by overall movement of CSF

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What are the functions of the CSF? (1)

Excretion/drainage and clearance of toxic metabolites, large molecules and drugs (acts as lymphatic system for brain clearing). Provides buoyancy to brain, reduces crushing spinal nerves, reduces brain weight (brain weight reduced from 1400 g to 50 g).

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What are the functions of the CSF? (2)

Distributes nutrients to brain – vitamin C, folate, riboflavin (those which have no efflux transporters via BBB). Distributes hormone secretions (IGF-2, leptin) through CNS

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Describe features of drug clearance by CSF (1)

CSF flow is pulsatile, helps clearance. CSF velocity varies with cardiac cycle. Pulses mix fluid back and forth through ventricles, sub-arachnoid space and peri-vascular space to help brain clearance

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Describe features of drug clearance by CSF (2)

Movement towards to the posterior end of the brain then towards anterior side of the brain – in time with the cardiac cycle (CSF movement backwards and forwards). Blood pressure pushes on lateral ventricles, synchronised with cardiac cycle

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Describe features of drug clearance by CSF (3)

Increase in blood pressure, increase in blood flows in arterioles . CSF is not steady and slow – pulses mix fluid back and forth through ventricles, sub-arachnoid space and peri-vascular space – helps brain clearance. (clinical links to dementia)

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Describe features of drug clearance by CSF (4)

CSF flow is circadian, drug is cleared at night (need to sleep for the CSF to remove toxins from the brain). Peak production (midnight peak - 42 ml/h, mid-day minimum production - 8ml/hr). Natural sleep/anaesthesia increases clearance of drugs and metabol

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Describe features of drug clearance by CSF (5)

Clearance of drugs from CSF – nasal route and sub arachnoid space. Small molecules removed from the brain quickly. Larger molecule in the brain – might stay there for longer

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Clearance depends on which two drug factors?

Clearance of drug depends on – size (larger molecular takes longer to clear), whether it is taken up by cells/binds to neurones (less likely to be removed away by the CSF)

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What are the obstacles to drug delivery to the brain?

Physical barrier (<400 Da, log P > -1). Transport barrier (active efflux transporters). Metabolic barrier (CYP450 enzymes metabolize small, permeant drugs). CSF clearance (drugs removed from brain fluids, faster at night). Problems for biologics and chemo

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What are the problems with current drugs to treat brain cancer? (1)

Primary brain tumours – very aggressive, gliomas, astrocytes etc. Start with core tumour, radiates out (metastases) – need drug treatment, not really suitable for surgery. Metastases from peripheral tumours – skin, breast, prostate cancer

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What are the problems with current drugs to treat brain cancer? (2)

Tumour molecular targets – inhibit glioma growth and proliferation, growth factor receptors, intracellular signaling pathways to cause apoptosis, block cell division/prevent mitosis/inhibit DNA replication.

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What are the problems with current drugs to treat brain cancer? (3)

Targets are beneficial in systemic treatment but failed in treatment of glioma

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What are the problems with current drugs to treat brain cancer? (4)

Gene modifiers (e.g. siRNA, cDNA, increase p53 for apoptosis, knockdown growth factor to slow tumour growth)– large hydrophilic drugs (cannot pass physical barrier), RNA/DNA degraded by nucleases (metabolic barrier), BBB actively degrades free nucleic aci

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What are the problems with current drugs to treat brain cancer? (5)

Chemotherapeutics (prevent cell division/DNA synthesis e.g. vincristine, vinblastine, paclitaxel, doxorubicin, daunorubicin, irinotecan) – large lipophilic drugs, stopped by transport barrier/efflux transporters P-gp, BCRP, MRP

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Chemotherapeutics need to overcome which two problems?

Efflux transporters at the BBB and efflux transporters at the brain-tumour cell barrier (BCRP, MRP, P-gp, substates, diagram)

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What are the advantages of using nanoparticles to deliver drugs across the BBB?

Encapsulate drug to: Protect it from Metabolic Barrier and efflux Transport Barrier. Carry a relatively large drug cargo to brain. Can carry almost any type of drug. High molecular weight drugs delivered

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What are the disadvantages of using nanoparticles to deliver drugs across the BBB?

Toxicity of the nanoparticle itself. Accumulation of nanoparticle by liver and spleen. Does not only target brain (will enter other organs too). Clinical Trials needed for both the therapeutic drug AND the nanoparticle itself, make trials costly and time

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What are the major types of nanoparticles? (1)

Polymer type – polyesters, amino acids, simple, cheap to make, first to the used with drug absorbed on the outside, some in central area. Carbon nanotubes – at early stage. Inorganic nanomaterials – gold, used for imaging not treatment.

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What are the major types of nanoparticles? (2)

Liposome – lipid bilayer, most commonly used, most tolerated. Changing nanoparticle features can enhance function.

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How can nanoparticles be optimized (composition)? (1)

Synthetic (cleared by kidney/liver quickly). Polymers (ease of manufacture, few interaction sites/drug loading areas). Dendrimers (large SA for drug attachment).

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How can nanoparticles be optimized (composition)? (2)

Natural composition – more tolerated. Avoid immune system, liver and kidney clearance. Phospholipids (normal cell walls). Liposomes (good, flexible drug loading), lipid soluble drug in shell, hydrophilic drug in core, but large NP size,

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How can nanoparticles be optimized (composition)? (3)

Lipophilic drugs – on the outside. Hydrophilic drugs – on the inside. Issue – liposome can increase in size, induce inflammation, not broken down (dense core of particles). Inorganic (stable easy to manufacture, but S/E - disturb BBB structure, induce inf

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How can nanoparticles be optimized (size and shape)?

Best size for penetration is 50-100 nm. NPs <20 nm, they are cleared quickly by the kidney, limited drug capacity, quick drug release. Spherical NPs easiest to prepare. Nanorods have higher brain accumulation (200 nm rapidly cleared by liver, 500 nm rods

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How can nanoparticles be optimized (charge)?

Neutral/negatively charged NPs (stable, retain drug, long blood circulation half-life, low rate of non-specific cellular uptake). Positively charged NPs (more easily penetrate cell membrane which have negative charge but more toxic). Neutral/negative NPs

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How can nanoparticles be optimized (protein adsorption)?

Protein adsorbs rapidly onto NP in blood. Accelerated clearance by white blood cells (RES). Tendency for protein to coat NP – phagocytosis of NP (add PEG coating to, minimize protein fouling, improves stability in blood, decrease clearance, increase bioco

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How can nanoparticles be optimized (BBB interaction)?

Ligands added to improve BBB targetting and transport

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Give an example of non-BBB targeting NP

BionTech/Pfizer SARS-CoV-2 vaccine. Lipid NP containing mRNA for virus spike protein. Drug delivery in the periphery. Lipid nanoparticle - cationic lipid, phosphatidylcholine / cholesterol (biomimetic), PEG (slows down the clearance of NPs), diameter ~ 80

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Give examples of NPs formulations being trialed for brain tumour

Onivyde MM-398: Liposome NP + PEG, 100nm diameter, Drug cargo: Irinotecan, First use, pancreatic cancer. Caelyx / Myocet: Liposome NP + PEG, 180nm diameter, Drug cargo: Doxorubicin, First use, ovarian cancer

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How can NPs be optimized for brain delivery?

Trojan horse technology. Attach molecules to the NP that are recognized by BBB uptake transporters. Trick BBB into taking up the whole BP

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What is the main approach for using Trojan Horse drugs (using ligands for BBB receptor-mediated transcytosis uptake)? (1)

Use an endogenous molecule on the surface of a nanoparticle that the BBB can recognise and will transport. This is the ‘Trojan’ ligand. The BBB will transport the Trojan ligands with the attached nanoparticle and drug cargo into brain.

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What is the main approach for using Trojan Horse drugs (using ligands for BBB receptor-mediated transcytosis uptake)? (2)

The most successful uptake mechanism targeted is Receptor-Mediated Transcytosis (RMT). Transcytosis can deal with the large size of the NP plus cargo drug

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Describe features of RMT (1)

Specific uptake of larger molecules. TfR, InsR, LRP, cytokines, viruses. Formation of transcytosis vesicle from Clathrin lattice. AP2 brings lattice down, pinched off by proteins, vesicle formed. Clathrin on outer side, AP2 in the middle, inside has lipid

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Describe features of RMT (2)

Molecules are well protected from metabolic barrier. Transcytosis avoids metabolic barrier and goes around the physical barrier

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Give examples of BBB receptors that can initiate RMT

Transferrin (iron carrying), LRP (transport lipoproteins), InSR (moving insulin to the brain) etc (diagram). Non of these transport systems are unique to BBB. But the BBB has high levels of TfR, LRP which are most often used by Pharma.

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Give an example of using a transferrin receptor Trojan ligand (1)

Nanoparticle delivers DNA (p53 gene plasmid) to brain, to stop cell division. Drug (plasmid encoding p53 gene, expression of p53 induced cell cycle arrest and apoptosis). Composition (liposone, neutral charge).

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Give an example of using a transferrin receptor Trojan ligand (2)

Size (100 nm diameter, optimise cell uptake). BBB Trojan (targets transferrin RMT, use ligand TfRsc, anti-transferrin receptor short chain). NP cross BBB by RMT using transferrin receptor. Enters glioma cell by endocytosis. NP ruptures in acid late endoso

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Give an example of using a transferrin receptor Trojan ligand (3)

Releases drug (p53 plasmid, stops cell division and causes apoptosis). Tumour cell (overexpresses receptor). Clinical trial SGT-53. Pre-clinical studies – p53 expressed in glioma cells but doesn’t last (at 72 hours), not a sustained increase in p53

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Give an example of using a transferrin receptor Trojan ligand (4)

Glioma tumour volume slowed down by p53 but then tumour volume increases, not sustained. NP gets across BBB, delivers drug to tumour cells (p53 plasmid) but this treatment alone is not enough to stop the tumour from growing

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Give an example of using an LRP Trojan ligand (1)

Nanoparticle to deliver Chemotherapy Paclitaxel (PTX) to brain. Drug (paclitaxel PTX enclosed inside NP). Composition (made from PEG, increases circulation time from <5h to 24h). Size (50-80 nm diameter, optimise cell uptake). BBB Trojan targets LRP RMT u

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Give an example of using an LRP Trojan ligand (2)

Distribution of NP – more in brain and liver, less in spleen and lung. Liver has lots of LRP receptors – movement of lipoproteins, liver involved in regulation of lipoproteins. Brain tumour size/volume reduced most with ANG-NP (14 day treatment), life exp

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Give an example of using an LRP Trojan ligand (3)

NP gets across BBB, delivers drug to tumour cells, more effective than giving drug alone

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Give an example of a NP taken up by two BBB uptake systems (1)

PEG - Liposome Nanoparticle, Using LRP ‘Trojan’ ligand, ANG. NP taken up by RMT using ANG (LRP Trojan). NP taken up by AMT (PEG removed by endothelial enzymes MMPs, charge of NP changes from -7mV to +2mV, positive charge surrounded by neutral PEG, link wi

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Give an example of an NP taken up by two BBB uptake systems (2)

NP approaches BBB, NP uses metabolic barrier to remove PEG (proteases break down peptide bonds in PEG) and increase uptake, nanoparticle becomes positively charged, AMT. More uptake of nanoparticle in cell culture tumour cells when using RMT and AMT toget

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Give an example of an NP that can move (chemotaxis) - 1

Use glucose metabolism to generate an oxygen jet. Polymersome – contains enzymes to oxidise glucose, glucose oxidase, catalase, to produce oxygen. Build-up of oxygen within a NP, needs to come out .

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Give an example of an NP that can move (chemotaxis) - 2

Asymmetric polymersome – area made thinner so oxygen can escape. Oxygen escapes through thin wall, propels NP backwards, aids movement across BBB due to moving about constantly. Uptake into the brain is fast. NPs enter neurones/glia, overcomes problem of

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What are biologics?

Drugs with biological origins e.g. peptides, nucleic-acid based compounds, cytokines, replacement enzymes, recombinant proteins, mAb

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What are the advantages of biologics? (1)

Highly specific targeting of a single aspect of complex diseases e.g. reducing amyloid peptide production, reducing Tau neurofibrillary tangle formation, inhibit neuronal death by apoptosis, or glutamate toxicity, delivering supportive growth factors.

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What are the advantages of biologics? (2)

Specific, metabolized by specific enzyme (not hepatic). Few (relatively) unwanted effects elsewhere in the body

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What is the disadvantage of biologics?

Antibodies are very large molecules (≈150,000 Da), very hard to deliver to brain, unstable, low permeability

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What are the general characteristics of small molecules?

Low MW (<1kDa), stable, simple, non-specific, oral ROA, high permeability, hepatic metabolism, easily distributed via circulation, no immunogenicitiy

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What are the general characteristics of biologics?

Large MW (1-220 kDa), unstable, complex structure, specific, parenteral, invasive ROA, low permeability, no hepatic metabolism, distributed via circulation/lymphatics (limited). Immunogenicity. mAb widely used to treat AD.

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Describe the use of mAb in the treatment of AD

Clinical trials – largely negative on primary and secondary outcome variables. Adverse effects – amyloid-related imaging abnormalities, BBB and tissue damage. 3 on-going trials – focus on pre-symptomatic people with a risk of AD. Aim is to slow down progr

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Why is there a lack of success in the use of antibodies to treat AD?

Poor antibody transport across BBB. Antibody transport across BBB is <0.1% (insufficient for therapeutic effect) due to: large, hydrophilic, no specific transporter (need to use Trojan antibodies)

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Describe features of Trojan antibodies (1)

Used to cross BBB via RMR. Antibodies must be raised against a receptor for RMT e.g. InR (insulin), TfR (transferrin). These Trojan antibodies are recognised as a natural ligand by a RMT receptor

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Describe features of Trojan antibodies (2)

Not all RMT transport systems can be used. Using InsR RMT Trojan antibody may disrupt glucose control throughout the body/organ damage. Choice of RMT target important to prevent unwanted effects.

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Describe features of Trojan antibodies (3)

Using Trojan antibody for RMT transport allows more antibody into the brain – but not yet a therapeutic antibody

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How nanoparticle and antibody therapies can be optimized for brain delivery and overcome the main problems of the BBB?

Overcome problem of antibody sticking to BBB capillaries. Combine a therapeutic antibody and Trojan antibody (3 examples)

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Describe how to overcome the problem of antibody sticking to BBB capillaries (1)

Trojan antibodies have very high affinity for RMT receptor, targets RMT at BBB very well, but affinity is too tight, antibody is not released to brain side after transport, Trojan antibody builds up inside the capillary endothelial cells – special problem

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Describe how to overcome the problem of antibody sticking to BBB capillaries (2)

A special problem using antibodies because they are capable of very high-affinity binding. Solution – reduce affinity of Trojan antibody for the BBB receptor

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Describe how to overcome the problem of antibody sticking to BBB capillaries (3)

Reduce affinity enough so Trojan antibody can be released from transcytosis vesicle after transport across BBB and get into the brain. But not too much or antibody will not bind to RMT receptor. Optimised affinity allows antibody entry to brain while stil

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Describe how to overcome the problem of antibody sticking to BBB capillaries (4)

Increases brain uptake but not yet a therapeutic antibody

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Describe the process of combining a therapeutic antibody and Trojan antibody - bispecific antibody? (1)

One side is a Trojan antibody to target TfR, other side is therapeutic antibody to inhibit amyloid beta-protein (anti BACE1), conjugation, half (get into brain), half (therapeutic effect) – increased brain uptake.

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Describe the process of combining a therapeutic antibody and Trojan antibody - bispecific antibody? (2)

Trojan part of antibody works – more bispecific antibody in brain after 12 hours compared to plan therapy antibody. Therapeutic part works – reduced amyloid beta production in the brain after 24 hours, inhibition of BACE-1 enzyme and amyloid synthesis

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Describe the process of combining a therapeutic antibody and Trojan antibody - the brain shuttle? (1)

Start with two different antibodies, Trojan antibody and therapeutic antibody, take binding fragments (Fab) and fused them to the therapeutic antibody to form a large double Ab (dFab) and also formed a sFab

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Describe the process of combining a therapeutic antibody and Trojan antibody - the brain shuttle? (2)

Increased single sFab in brain compared to therapeutic antibody after 24 hours, better uptake than dFab and fewer amyloid beta plaques formed after 4 months treatment of sFab, prevents formation of plaques and removes old plaques from the brain

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Describe the process of combining a therapeutic antibody and Trojan antibody - the brain shuttle? (3)

dFab antibody stuck in endocytosis vesicles – similar to high affinity antibodies. sFab antibody transported to brain – similar to low affinity antibodies

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Describe the process of combining a therapeutic antibody and Trojan antibody - ATV (1)

The antibody transport vehicle ATV – engineer TfR binding site into heavy chain Fc of a general antibody, replace general antibody Fabs with therapeutic antibody Fabs.

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Describe the process of combining a therapeutic antibody and Trojan antibody - ATV (2)

General antibody and Trojan binding site, engineered TfR binding site, take antigen binding site of therapeutic antibody (anti-BACE 1), combine parts together

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Describe the process of combining a therapeutic antibody and Trojan antibody - ATV (3)

ATV – therapeutic antibody Fabs + heavy chain with TfR binding site. Reduces amyloid in brain after 4 weeks, inhibits amyloid synthesis by binding to BACE-1. A very flexible approach, can add different biologics to the TfR Fc.

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Describe the process of combining a therapeutic antibody and Trojan antibody - ATV (4)

Creation of two therapeutic antibodies with 2 different Fabs – ATV anti-amyloid/anti-Tau. Other biologics – peptides, enzymes, iduronate 2-sulfatase, lysosome enzyme

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Drug Delivery Across the Blood Brain Barrier

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