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Macromolecules in drug delivery
Macromolecular targeting agents, carriers, and drugs
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Why macromolecules in drug delivery?
WHY?
Increase therapeutic index
Reduced side‐effects
Prolonged effects
HOW?
Targeting
A carrier for small drugs
A release mechanism (if necessary)
Protection of drug cargo
Classic chemotherapy
Drug delivery using macromolecular system
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How?
Overcome obstacles
Capitalize on opportunities
Classic chemotherapy
Drug delivery using macromolecular system
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Blood flow through the body
Bertrand and Leroux. J Controlled Release (2012) 152–163
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“Physical” obstacles to drug delivery
Pulmonary capillaries
(2–13 µm)
Pore size 4.2 nm
Bertrand and Leroux. J Controlled Release (2012) 152–163
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“Chemical” obstacles to drug delivery
Spleen or Liver
Marasco and Sui. Nature Biotechnology (2007) 25, 1421–1434
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Passive targeting – the EPR effect
Enhanced Permeation and Retention
Kratz et al. ChemMedChem 3 (2008) 20-53
Khandare and Minko. Prog Polym Sci 31 (2006) 359-397
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Active targeting
Peer, Karp, Hong, Farokhzad, Margalit, Langer. Nature Nanotechnology (2007) 2, 751–760
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Outline
1. Macromolecular targeting agents + carriers
* Release of drug via a linker
Drug
2. Macromolecular carriers
* Small molecule targeting ligands
3. Macromolecular drugs
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SECTION 1 – Antibody–drug conjugates
Antibodies are examples of carriers which are simultaneously targeting ligands
 Anticancer drugs are given at sub‐optimal doses because of toxicity to healthy tissue.
 Adding a targeting moiety increases the dose of drug to the target tissue.
 Important issues:
 Choice of targeting ligand
 Choice of drug
 Choice of linker
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Zolot, Basu, Million, Nature Reviews Drug Discovery (2013) 12, 259–260
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Choice of target
Receptor expression:
 High density on surface of target
 For example, a receptor density of 105 ERBB2 receptors per cell was required for an improved therapeutic effect of anti‐ERBB2‐targeted liposomal doxorubicin over non‐targeted liposomal doxorubicin in a metastatic breast cancer model
 High degree of homogeneity
 Must not be shed by cells
 Circulating shed antigen will compete with the target cells for binding of the targeted therapeutics, and any complexes that form would be rapidly cleared from the circulation
Allen. Nature Rev Cancer 2 (2002) 750-763
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Consequence of ligand–receptor binding
Binding of antibodies to their receptors can cause internalization, but not always.
Allen. Nature Rev Cancer 2 (2002) 750-763
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A variety of targets expressed by tumors
Schrama et al. Nature Rev Drug Disc (2006) 5, 147–159
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Issues for targeting
 Binding affinity (high or low)?
 Tumor (relatively low binding)
 In circulation (relatively high binding)
 EPR effect helps concentrate antibodies in tumors
 Distribution to target location (linker must be stable)
Allen. Nature Rev Cancer (2002) 2, 750–763
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Choice of drug
 Antibodies have low drug loadings
 Large amounts of antibodies can saturate the binding site
 Expensive
 Increased chance of immunological reactions
 The drug must be very potent
 e.g., calicheamicin, the maytansine derivative DM1, or monomethyl
auristatin E

Drug loading must be low to permit FcRn recycling
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FcRn recycling
Roopenian, Akilesh. Nature Reviews Immunology (2007) 7, 715–725
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Immune response to antibodies
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How to attach drugs to antibodies
Garnett. Adv Drug Del Rev 53 (2001) 171-216
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Drug release – Linker chemistry
Extracellular enzymes
Redox
Enzymes, pH, redox
Allen. Nature Rev Cancer 2 (2002) 750-763
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Linker considerations
Permanent bonds
Useful if the carrier is (bio)degradable
Risk of drug inactivation
Labile bonds
Risk of pre-mature release
Used to trigger drug release upon internalization
The choice of linker is crucial for the success of the drug delivery system.
Macromolecule
Labile bonds
—Linker—Drug
Permanent bond
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Common “permanent” linker chemistry
A
+
B
=
Amide
Amine
Carboxylic
acid
Amine (via Schiff base)
Amine
Thioethers
Thiol
R: Drug ; Prot: Macromolecule
Gauthier and Klok. Chem Commun (2008) 2591-2611
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“Labile” linker chemistry
Type
Structure
Use/ Comments
Redox-sensitive
R–SS–R (disulfides)
Reduction in the
cytoplasm releases the
drug
Relatively unstable in the
blood
pH-sensitive
—N=N–C (hydrazones )
Orthoesters
Acetals, etc.
Designed to cleave in the
acidic
endosomal/lysosomal
compartments
Enzyme sensitive
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Enzyme-cleavable linkers
These are all short peptides easily prepared synthetically
Kratz et al. ChemMedChem 3 (2008) 20-53
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Some considerations
Location of cleavage is not as well established as for redox- or pH-sensitive linkers
Kratz et al. ChemMedChem 3 (2008) 20-53
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Example : Mylotarg
Mylotarg, a conjugate of the cytotoxic antibiotic calicheamicin and an antiCD33 humanized antibody, is the only antibody prodrug that has received
market approval… but …
Kratz et al. ChemMedChem 3 (2008) 20-53
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Some concluding remarks
 Low drug loading (3–10 molecules per antibody)
 Pre-clinical studies often show:
 Better accumulation in tumors versus non-targeted
antibodies
 Better efficacy versus free drug
 Reduced toxicity versus free drug
 This approach is well-suited to very cytotoxic drugs
 Immunogenicity: requires chimeric or humanized abs.
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Outlook
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SECTION 2 – Macromolecular carriers
Polymer therapeutic are examples of inert carriers
with pendant targeting units and drugs
Small drugs distribute randomly through the body.
This often leads to side effects.
The attachment to a polymeric carrier can:
Prolong circulation lifetime to promote passive tumor uptake.
Promote targeting and receptor-mediated uptake through a targeting ligand.
Duncan Nature Rev Cancer 6 (2006) 688-701
Ringsdorf. J Pharm Sci Polym Sci 51 (1975) 135-153
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Targeting polymer therapeutics
Passive targeting via EPR effect
Active targeting via a targeting ligand
Kratz et al. ChemMedChem 3 (2008) 20-53
Khandare and Minko. Prog Polym Sci 31 (2006) 359-397
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Basic requirements for the carrier
 The polymer must be non-toxic and non-immunogenic
 Polymer MW should be high enough to ensure long circulation, but nonbiodegradable polymers must be less than 40,000 g.mol-1 to enable renal
elimination.
 Usually MW is between 30,000 – 100,000 g.mol-1
 The polymer must be able to carry an adequate payload
 The polymer must be stable during transport, but release the drug upon
arrival at target location
Duncan Nature Rev Cancer 6 (2006) 688-701
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Types of carriers
Carrier
Example
Proteins
Antibody, antibody fragment
Albumin
Lectins
Hormones (peptides), etc.
Uni-molecular
Can also be used as targeting ligands
Dextran
Hyaluronic acid
Heparin sulfate
Natural biopolymers
Variable structure
Polysaccharides
Synthetic
polymers
Poly(L-lysine), poly(L-lysine citramide)
Poly(L-glutamic acid)
Poly(α,β-(N-hydroxyethyl)-D,L-aspartamide)
Poly(N-(2-hydroxyethyl)-L-glutamine)
Poly(L-aspartic acid)
Poly(N-(2-hydroxypropyl)methacrylamide)
Poly(ethylene glycol)
Poly(styrene-co-maleic acid) (SMA)
Dendrimers, hyperbranched polymers, etc.
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Polydisperse
Flexibility of design
Cheap and easy to prepare
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Most commonly used synthetic polymers
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PEGylation of small-molecule drugs
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 Enhances water solubility and decreases immunogenicity
Limited conjugation capacity (only two terminal functional groups exist at the end of the
polymer chain)
This limitation can be overcome by use of a multi-functional linker (but can lead to steric
hindrance problems)
Loss of activity (mMEG5000 pactilaxel-7-carbamates 103 times less active than native drug)
Large PEG blocks the activity at the target cell
Drug does not reach target cells in sufficient concentration (low loading)
Greenwald et al. Adv Drug Del Rev 55 (2003) 217-250
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Poly(glutamic acid) (PGA)
Random-coil at pH 7
High drug loading
Biodegradable (cathepsin B, found in mice not expressing this enzyme)
Slow release of drugs by hydrolysis and fast release by enzymatic degradation
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PGA conjugates
PGA–paclitaxel conjugate (CT-2103; Xyotax)
In phase I/II clinical trials, PGA–paclitaxel (CT-2103) showed a significant number of partial responses or
stable disease (patients with mesothelioma, renal cell carcinoma, non-small cell lung carcinoma (NSCLC))
ovarian cancer.
In one recent randomized phase III clinical trial, PGA–paclitaxel was compared with gemcitabine or
vinorelbine as a first-line treatment for poor performance status (PS2) NSCLC patients. The conjugate showed
significantly reduced severe side effects when compared with control patients, most of whom received
gemcitabine.
A PGA conjugate (CT-2106) of Mw 50,000 g mol-1 and containing a Gly linker to camptothecin (33–35 wt%)
has also entered phase I/II trials.
Duncan Nature Rev Cancer 6 (2006) 688-701
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Poly(2-hydroxypropyl methacrylamide) (pHPMA)
Since 1973, PHPMA is the most investigated and advanced polymer used
in therapeutics due to its versatility as a vehicle.
Can be modified along its side chain
PHPMA being hydrophilic, increases water solubility of the drugs and has
proven to be non-toxic in rats.
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Synthesis of pHPMA
Two possible routes for preparing HPMA-drug conjugates
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Bottom-up synthesis
Lu et al. J Controlled Release 78 (2002) 165-173
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Post-polymerization modification approach
Active ester group for post-polymerization
modification with a targeting moiety
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Example of a pHPMA conjugate
A PHPMA copolymer with adriamycin conjugated with the peptidyl linker Gly-Phe-Leu-Gly (PK1), has been
developed. These conjugates are less toxic than the free drug and can accumulate inside solid tumor
models.
An HPMA copolymer–Gly-Phe-Leu-Gly-doxorubicin conjugate that also contained galactosamine (PK2;
FCE28069) was designed to promote multivalent targeting of the hepatocyte asialoglycoprotein receptor
(ASGR) to treat primary liver cancer.
Duncan Nature Rev Cancer 6 (2006) 688-701
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Dextran
 Dextran is poly(glucose) with α-1,6 linkages.
 Multiple groups for drug conjugation.
 Conjugation can also be achieved by prior periodate oxidation of the polymer
 After oral administration, the polymer is not significantly absorbed.
 Effective applications of dextran as polymeric carriers are through injections.
41 53 (2001) 171-216
Garnett. Adv Drug Del Rev
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Example of dextran–drug conjugate
K1, K2 : slow hydrolysis
K3 : enzymatic hydrolysis
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Prolonged circulation
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Example of targeted dextran–doxorubicin
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Importance (effect) of drug loading
Too high loading can lead to a significant change in the
properties of the drug delivery system!
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Antibody vs. non-antibody targeting agents
Readily available,
inexpensive, and easy to
handle, some selectivity
Very specific,
expensive, timeconsuming to produce,
immunogenicity, etc.
Allen. Nature Rev Cancer (2002) 2, 750–763
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Targeting ligands
Folate receptor over-expressed in many human cancers.
Large amounts of folic acid in food competitively reduces the efficiency of this ligand
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Cell penetrating peptides
Short peptides of less than 30 amino acids that are able to penetrate cell
membranes
They translocate different cargoes into cells.
They are amphipathic and net positively charged.
The mechanism of cell translocation is not known but it is apparently receptor
and energy independent although, in certain cases, translocation can be
partially mediated by endocytosis.
Cargoes that are successfully internalized by CPPs range from small
molecules to proteins and supramolecular particles.
Most CPPs are inert or have very limited side effects.
Zorko and Langel. Adv Drug Del Rev 57 (2005) 529-545
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Examples
Zorko and Langel. Adv Drug Del Rev 57 (2005) 529-545
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In clinical trials
Kratz et al. ChemMedChem 3 (2008) 20-53
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Some conclusions
 Natural or synthetic macromolecular carriers offer a large amount of
flexibility for conjugate design.
 They constitute excellent means for altering the biological properties of
small-molecule therapeutics.
 High drug loading (beware steric hindrance and spacer length)
 Possibility of adding targeting ligands
 Interesting for combination therapy (more than one type of drug attached to
the polymer
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SECTION 3 – Macromolecular drugs
Therapeutic proteins are examples of carriers which are simultaneously drugs
 Proteins that are engineered in the laboratory for pharmaceutical use are
known as therapeutic proteins.
 The majority of biopharmaceuticals marketed to date are recombinant
therapeutic protein drugs.
 Today therapeutic proteins are used to relieve patients suffering from
many conditions, including:
 Various cancers (Monoclonal antibodies, Interferons) , Heart attacks, strokes, cystic
fibrosis, Gaucher's disease (Enzymes, Blood factors), Diabetes (Insulin), Anaemia
(Erythropoietin), Haemophilia (Blood clotting factors)
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Obstacles to protein delivery
Frokjaer and Otzen. Nature Rev Drug Disc 4 (2005) 298-306
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Other obstacles
Aggregation
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Other obstacles
Immunogenicity
Must produce highly pure proteins that are (nearly) identical to human proteins to
avoid T and B lymphocytes
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Protein engineering approach
Example of insulin
Mutation of amino acid
sequence to promote
monomer formation
(fast release)
Chemical attachment of fatty
acids to the protein surface
can increase affinity to serum
albumin. This can increase its
circulation time in the blood
(slow release)
Frokjaer and Otzen. Nature Rev Drug Disc 4 (2005) 298-306
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Chemical engineering approach – PEGylation
Why PEG?
Non-toxic and non-immunogenic
Flexible, highly water soluble, has a hydrodynamic radius which is ca 5-10
times greater than that of an equivalent globular protein
Can be prepared with very specific ligation chemistry
Veronese and Pasut. Drug Delivery Today 10 (2005) 1451-1458
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Some approaches for PEGylation
Veronese and Pasut. Drug Delivery Today 10 (2005) 1451-1458
Gauthier and Klok. Chem Commun
(2008) 2591-2611
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Example – L-asparaginase
Gauthier and Klok Polym Chem (2010) 10.1039/c0py90001j
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Effect of PEG MW on renal clearance
Luxon et al. Clin Ther 24 (2002) 1363-1383
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Circulation half-life
Luxon et al. Clin Ther 24 (2002) 1363-1383
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Maintaining protein structure
Permanent bond between PEG and INF!
PEG
Loss of ~93 % of antiviral activity of IFN
In this case, activity not related to PEG
MW.
This can contrast to when PEG is added
to amino groups on this protein
Interferon α-2b
Shaunak et al. Nature Chem Biol 2 (2006) 312-313
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Some conclusions
 PEGylation is an effective means of improving the properties of therapeutic
proteins (circulation time, immunogenicity, etc.)
Limitations:
 PEG is not unimolecular (potentially different biological properties)
 This leads to a population of drug conjugates, which might have different biological properties,
mainly in body-residence time and immunogenicity.
 This is important for low MW proteins
 the polydispersity problem must be taken into consideration when dealing with low molecular-weight
drugs, either peptide or non-peptide drugs, where the mass of linked PEG is more relevant for
conveying the conjugate's characteristics (size).
 Large MW PEG is not excreted
 As for other polymers, PEGs are usually excreted in urine or feces but at high molecular weights
they can accumulate in the liver, leading to macromolecular syndrome.
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Accepted by US FDA
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