Peptide and CNS drug design exam
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What cells form the blood-brain barrier (BBB)?
A single layer of capillary endothelial cells, surrounded by pericytes and astrocytes.
What structures seal adjacent BBB endothelial cells?
Tight junctions between capillary endothelial cells.
Name two proteins found in BBB tight junctions.
Occludin and claudins.
How does BBB tightness compare with the intestinal barrier?
BBB tight junctions are tighter and restrict drug entry into the brain.
Which neurological conditions are associated with BBB dysfunction?
Stroke, neurodegenerative diseases, immune disorders (e.g. MS), and brain tumors.
What is the brain-to-plasma ratio, Kp?
Kp = Cb/Cp, where Cb is total brain concentration and Cp is total plasma concentration.
What does the free drug hypothesis state?
Only a fraction of the drug is unbound and free in plasma (fu,p) or tissue (fu,b for brain) to diffuse across biological barriers
What do fu,p and fu,b represent?
fu,p is the unbound fraction in plasma; fu,b is the unbound fraction in brain tissue.
What is Kp,uu?
= Cb,u/Cp,u; The ratio of unbound brain concentration to unbound plasma concentration
Why can Kp and Kp,uu differ substantially?
Kp measures total concentrations, whereas Kp,uu accounts for the unbound fractions in plasma and brain.
What does Kp,uu approximately equal to 1 indicate?
Passive transport/equilibrium across the BBB.
What does Kp,uu significantly below 1 indicate?
Net active efflux across the BBB.
What does Kp,uu significantly above 1 indicate?
Net active uptake/transport into the brain.
Which brain compartments are shown in the BBB concentration schematic?
Interstitial fluid (ISF), intracellular fluid (ICF), and cerebrospinal fluid (CSF).
Why may total brain concentration fail to predict CNS pharmacology?
Total brain concentration does not necessarily reflect unbound drug concentration at the relevant site, such as ISF.
Approximately what percentage of drugs cross the BBB?
Less than 2%.
What is the principal passive-diffusion route across the BBB?
Transcellular diffusion through endothelial cells.
How do physicochemical requirements for brain penetration compare with oral absorption?
Brain penetration generally requires more restrictive physicochemical properties.
Which two small-molecule properties are most critical for BBB penetration?
Molecular weight (MW) and hydrogen-bond donors (HBD).
What CNS-critical molecular-weight guideline is given for small molecules?
MW ≤ 470 Da.
What CNS-critical TPSA guideline is given for small molecules?
Topological polar surface area (TPSA) ≤ 90 Ų.
What CNS-critical HBD guideline is given for small molecules?
HBD ≤ 2.
In the brain-exposure chart, what trend occurs as HBD count increases?
The fraction of brain-penetrant compounds decreases markedly as HBD count rises.
What do BP and PR mean in the brain-exposure assessment figure?
brain-penetrant and peripherally restricted.
What transporter classes are present at both BBB and intestinal barriers?
Uptake/influx transporters and efflux transporters.
What do transport proteins, that both the BBB and intestine have, do?
Transport compounds into CNS (BBB) or into blood from intestine
What is the effect of BBB efflux proteins on CNS drug exposure?
They transport drugs out of the brain and can limit CNS penetration.
What is P-glycoprotein also called?
Pgp or multidrug resistance protein 1 (MDR1).
Why is P-glycoprotein important in BBB drug discovery?
It is the most studied BBB efflux protein and can strongly limit brain penetration.
Where is P-glycoprotein expressed?
At both the BBB and intestinal barrier.
How does mouse BBB P-glycoprotein expression compare with human expression?
Mouse BBB Pgp expression is 2.3-fold higher than in humans.
What does BCRP stand for?
Breast cancer resistance protein.
Where is BCRP expressed?
At both the BBB and intestinal barrier.
How does human BCRP expression in brain microvessels compare with Pgp?
It is expressed at comparable levels to Pgp in human brain microvessels.
How does human BCRP expression compare with mouse BCRP expression?
Human expression is 1.85-fold higher than mouse expression.
How does mouse BCRP expression compare with mouse Pgp?
Mouse BCRP expression is about one-third that of Pgp.
What is the probability of launch from Phase I entry for CNS drugs cited in the lecture?
3%, compared with 9% for oncology and 16% for anti-infective agents.
What Alzheimer’s drug-development example illustrates the high failure rate?
From 2002–2012, 244 drugs entered Alzheimer’s clinical trials and only one was FDA-approved (memantine).
What are two structural strategies to improve passive BBB diffusion?
Increase lipophilicity and reduce HBD/TPSA.
How can efflux-mediated restriction of CNS exposure be addressed?
Reduce interaction with P-glycoprotein or other efflux transporters.
What is MPO in CNS drug design?
Multiparameter optimization: scoring/optimizing multiple physicochemical properties associated with CNS exposure.
What are major challenges in CNS drug design beyond BBB penetration?
Incomplete disease biology, disease heterogeneity, identifying disease-modifying targets, limitations of animal models, and finding useful biomarkers.
Why is target identification difficult in neurodegenerative disease?
Disease biology may be incompletely understood, and symptoms can appear after extensive neuronal loss.
What is the distinction between disease-modifying and symptomatic treatment?
Disease-modifying treatment alters disease progression; symptomatic treatment alleviates symptoms without necessarily changing progression.
Which Alzheimer’s target categories were prominent in the slide’s clinical-trial chart?
Amyloid-beta, neurotransmitter receptors, tau, inflammation/immune targets, synaptic plasticity/neuroprotection, and metabolism/bioenergetics.
Which therapeutic strategies for Parkinson’s disease were listed?
Alpha-synuclein targeting, GLP-1 receptor agonists, kinase inhibitors, antioxidants, anti-inflammatory approaches, microbiome approaches, and cell therapy.
What are the broad categories of brain-delivery technologies?
Invasive and non-invasive approaches.
What are some non-invasive brain delivery technologies?
Biological mechanisms such as receptor or adsorptive mediated transcytosis, cell-mediated brain delivery, cell-penetrating peptides, and receptor-mediated BBB opening, as well as intranasal delivery
What is the paracellular BBB pathway?
Movement between adjacent endothelial cells through junctional spaces.
Which molecules can use the paracellular pathway under normal conditions?
Primarily small water-soluble molecules; the pathway is highly restricted by tight junctions.
Is passive diffusion transcellular or paracellular?
Transcellular.
What is transcytosis?
Transport across a cell through uptake on one side, intracellular trafficking, and release on the opposite side.
What are the three mediated-transcytosis mechanisms discussed?
Carrier-mediated, receptor-mediated, and absorptive-mediated transcytosis.
What is carrier-mediated transcytosis (CMT)?
Transport proteins that move essential nutrients across the BBB.
How does a carrier protein transport its substrate?
Substrate binding induces a conformational change in the carrier, moving the substrate across the membrane.
What does the large neutral amino acid transporter carry?
Phenylalanine and other large neutral amino acids.
What CNS drug uses a large neutral amino acid transporter?
L-DOPA.
Why can L-DOPA enter the brain when dopamine cannot?
L-DOPA resembles a transported neutral amino acid; after entering the brain it is decarboxylated to dopamine.
What limits the applicability of CMT for drug delivery?
Transporters generally accept only low-molecular-weight compounds structurally similar to their natural nutrient substrates.
What characteristics make a receptor useful for receptor-mediated transport (RMT)?
High BBB expression, preferably selective expression in brain microvasculature than in peripheral tissues or brain parenchyma, and efficient transcytosis.
Why is receptor expression in brain microvasculature preferred over peripheral tissues or brain parenchyma?
It can improve selectivity for transport across the BBB.
What species difference is noted for transferrin receptor expression?
Transferrin receptor expression in mouse brain microvasculature is 2.5-fold higher than in humans.
What limits the delivery capacity of RMT?
Capacity is limited by receptor expression, potentially restricting use to very potent drugs.
How might RMT drug delivery affect endogenous physiology?
It may interfere with transport or function of the receptor’s endogenous ligand.
What does brain parenchyma mean?
Functional brain tissue.
What are the most common receptor mediated transport targets?
Low-density lipoprotein receptors, transferrin receptor, ion channels, and integrins
What are LRP-1 and LRP-2?
Low-density lipoprotein receptor-related proteins 1 and 2, members of the LDL receptor family.
Where are LRP-1 and LRP-2 expressed?
Both are expressed in the CNS.
What is notable about LRP-1 expression in glioma?
LRP-1 is highly expressed on glioma cells.
How can LRP proteins support brain delivery?
They bind and endocytose a broad range of proteins, enabling cargo transport through transcytosis.
What is the normal function of the transferrin receptor (TfR)?
It mediates transport of transferrin, an iron-binding protein, and regulates iron uptake and homeostasis.
What does transferrin transport across the BBB?
Iron.
How were several TfR-targeting peptides identified?
By phage display.
Which ion channel receptor is targeted for CNS delivery?
The nicotinic acetylcholine receptor, a ligand-gated ion channel expressed in the CNS and peripheral nervous system.
Which other ion channel target is mentioned?
Potassium channels.
Which integrin is listed as an RMT target?
Alpha-v-beta-3 (αvβ3) integrin.
Which receptor does Angiopep-2 bind?
LRP-1.
How does Angiopep-2 facilitate cargo delivery?
Angiopep-2 binds LRP-1; the complex is internalized and cargo can be transported across the BBB.
What types of cargo have been attached to Angiopep-2?
Small molecules and peptides, including neurotensin; it has also been used to modify nanoparticles.
What is ANG1005?
An Angiopep-2–paclitaxel conjugate, also called paclitaxel trevatide.
Why may Angiopep-2 be useful for glioma targeting?
LRP-1 is overexpressed on glioma cell membranes.
What are two peptides targeting low-density lipoprotein related protein 1 (LRP-1)?
Angiopep-2 and aproprotein E derivatives
What does PTX stand for in the Angiopep-2 conjugate slides?
Paclitaxel.
What are some common linkers for peptide-drug conjugates?
Ester, amide, hydrazone, and disulfide
How many copies of drug can be attached in the illustrated Angiopep-2 conjugate design?
Three copies via the N-terminus and two lysine residues.
Name three peptides targeting transferrin-receptors
THR, CRT, HAI
How is the CRT peptide cyclized?
By a disulfide bond.
Why does CRT cyclization improve peptide stability?
It stabilizes the peptide to metabolism by proteases
Name 2 peptides that target ion channel receptors
RVG29 and apamin
What is RVG29?
A 29-amino-acid peptide sequence derived from rabies virus glycoprotein that targets the nicotinic acetylcholine receptor.
What has RVG29 been used to deliver?
Used for gene delivery
What is apamin?
A neurotoxin from bee venom that binds a potassium channel.
What is MiniAp-4?
A smaller lactam derivative of apamin that can deliver cargo across the BBB.
What peptide motif is used to target integrins?
RGD (Arg-Gly-Asp) peptides, typically as macrocyclic analogs.
What are common applications studied for RMT delivery?
Glioblastoma and other brain tumors, as well as neurodegenerative diseases.
What formulations are often combined with RMT peptides?
Nanoparticles and liposomes.
What is absorptive-mediated transcytosis (AMT)?
Uptake driven by electrostatic interaction of positively charged peptides with negatively charged cell-surface components, followed by endocytosis.
What negatively charged cell-surface components are mentioned in AMT?
Heparin proteoglycans.
What are cell-penetrating peptides (CPPs) generally like?
Highly basic, positively charged peptides.
What are some of the cell penetrating peptides that have been studied extensively?
Tat peptide, penetratin, polyarginine, SynB1