peptide delivery
PART 1: FOUNDATIONS – PEPTIDE AND PROTEIN STRUCTURE
Section 1: Learning Outcomes (Page 3)
By the end of this lecture, students should be able to:
Appraise the structure of peptides and proteins.
Explain the classes of proteins/biopharmaceuticals.
Discuss the formulation aspects of peptides and proteins.
Evaluate the newer forms of peptide/protein delivery.
Section 2: Defining Peptides and Proteins (Page 4)
Basic Building Blocks: Both peptides and proteins are polypeptides – polymers composed of individual amino acids linked together by amide bonds (also called peptide bonds).
The 20 Amino Acids: There are 20 standard proteinogenic amino acids, each with a unique side chain (R-group) that dictates its chemical properties (e.g., hydrophobic, hydrophilic, charged, polar).
Distinction by Size (a conventional, not absolute, rule):
Peptides: Typically contain fewer than 50 amino acids. They are smaller, often lack extensive folded structure, and are sometimes referred to as oligopeptides.
Proteins: Typically contain more than 50 amino acids, often hundreds. They usually fold into a specific three-dimensional (tertiary) structure that is essential for their biological function.
Note on Insulin: Insulin, composed of 51 amino acids (two chains, 21 and 30 AAs, linked by disulfide bridges), is conventionally classified as a peptide despite crossing the 50-AA threshold. This highlights that the distinction can be fuzzy and is often based on historical and functional context.
Image Description (Page 5): Likely shows the general structure of an amino acid: a central (alpha) carbon (Cα) bonded to four groups: an amino group (-NH₂), a carboxyl group (-COOH), a hydrogen atom (-H), and a variable side chain (R-group) . This R-group defines the amino acid's identity and chemical characteristics.
Section 3: Levels of Protein Structure (Page 6)
Proteins are organised into four hierarchical levels of structure:
Primary Structure:
Definition: The linear sequence of amino acids in the polypeptide chain, from the N-terminus (amino end) to the C-terminus (carboxyl end).
Significance: This sequence is genetically determined and dictates all higher levels of structure and function. A single amino acid change (mutation) can have profound effects (e.g., sickle cell anaemia).
Secondary Structure:
Definition: Local, regular, repeating folding patterns within the polypeptide backbone, stabilised primarily by hydrogen bonding between backbone carbonyl (C=O) and amine (N-H) groups.
Common Motifs:
Alpha-Helix (α-helix): A right-handed coiled structure.
Beta-Sheet (β-sheet): A structure where adjacent segments of the chain align side-by-side, forming a sheet held together by inter-strand hydrogen bonds.
Visual Cues: The slide mentions specific structures (Haemoglobin B chain – α, Immunoglobulin Fold – β, Lactate Dehydrogenase – mixed α/β), indicating that images of these proteins would highlight regions of α-helix (often depicted as coiled ribbons) and β-sheet (often depicted as flat arrows).
Image Description (Page 6): Shows three-dimensional ribbon diagrams of representative proteins:
Haemoglobin B Chain: A largely α-helical protein. The image would show a structure dominated by coiled ribbons (helices).
Immunoglobulin Fold: A β-sheet-rich domain characteristic of antibodies. The image would show a "sandwich" of flat, broad arrows representing anti-parallel β-strands.
Lactate Dehydrogenase: A typical enzyme with a mixed α/β structure. The image would show a central core of β-sheet (arrows) surrounded by α-helices (ribbons).
Tertiary Structure:
Definition: The overall three-dimensional folding of a single polypeptide chain, resulting from interactions between side chains (R-groups) . This includes hydrophobic interactions, ionic bonds (salt bridges), hydrogen bonds, and disulfide bridges (covalent).
Significance: Forms the functional protein, creating active sites, binding pockets, etc.
Quaternary Structure:
Definition: The arrangement of multiple polypeptide subunits (proteins) into a functional complex. Not all proteins have this level.
Example: Haemoglobin is a tetramer of two α-globin and two β-globin subunits.
Section 4: Characterising Protein Structure (Page 7)
To ensure quality and stability, proteins must be characterised at both primary and higher-order levels.
Primary Structure Characterisation:
Goal: Determine the amino acid sequence and identify any mutations or post-translational modifications.
Methods:
Amino Acid Sequencing (e.g., Edman degradation): A classic method that sequentially cleaves and identifies amino acids from the N-terminus. Destructive.
Digest Mapping + Mass Spectrometry (MS): The protein is digested with specific enzymes (e.g., trypsin) into smaller peptides. These peptides are separated (e.g., by HPLC) and their masses measured by MS. The resulting "peptide mass fingerprint" is compared to the theoretical sequence. Highly sensitive and accurate.
Secondary and Tertiary Structure Characterisation:
Goal: Assess the correct folding and conformation. Unfolding (denaturation) can indicate instability.
Method: Circular Dichroism (CD) Spectroscopy.
Principle: Optically active molecules (like the chiral backbone and aromatic side chains of proteins) absorb left- and right-circularly polarised light to different extents. The difference in absorption creates a CD signal.
Output: CD spectra in the far-UV region (190-250 nm) are characteristic of secondary structure:
α-helix: Strong double minima at 208 nm and 222 nm.
β-sheet: A single minimum around 216-218 nm.
Random coil: A minimum around 198-200 nm.
Advantages: Non-destructive, requires very little sample, and can be used to monitor structural changes over time (e.g., during stability studies).
Image Description (Page 7): Shows a typical CD spectrum overlay for poly-lysine in different conformations. One curve would have the characteristic double dip for α-helix, another a single dip for β-sheet, and a third a sharp dip at lower wavelength for random coil.
PART 2: CLASSES OF BIOPHARMACEUTICALS AND EXAMPLES
Section 5: Classes of Proteins/Biopharmaceuticals (Page 8)
This outlines the diverse range of therapeutic agents derived from biological sources or produced using biotechnology:
Recombinant Therapeutic Proteins: Produced by genetically engineered cells (bacteria, yeast, mammalian).
Examples: Human Growth Hormone (for growth disorders), Herceptin® (trastuzumab) – a monoclonal antibody for HER2+ breast cancer.
Peptides: Smaller polypeptides, often produced synthetically or recombinantly.
Examples: Insulin (diabetes), Calcitonin (osteoporosis), Goserelin (prostate/breast cancer), Oxytocin (labour induction).
Blood Products: Derived from human plasma.
Examples: Factor VIII (haemophilia A), Gamma globulin/Immunoglobulins (immune deficiencies), Serum Albumin (volume expansion).
Vaccines: Biological preparations that induce immunity.
Examples: Sub-unit vaccines (use specific peptide antigens), killed/inactivated whole organisms.
Nucleic Acids: Emerging class of therapeutics.
Example: Antisense oligonucleotides – short, synthetic strands of DNA/RNA designed to bind to and inhibit specific mRNA, preventing translation of a disease-causing protein.
Polysaccharides: Complex carbohydrates with therapeutic activity.
Example: Low Molecular Weight Heparin (LMWH) – an anticoagulant.
Image Description (Page 9): Shows images of three therapeutic proteins produced via biotechnology:
Insulin: Likely an image of insulin vials/pens or a molecular model.
Interferon β (Relapsing MS): A cytokine used to modulate the immune system in multiple sclerosis.
Interferon γ (Granulomatous Disease): Another cytokine used to treat chronic granulomatous disease by enhancing phagocyte function.
Bioreactor Image: A photo of stainless steel mammalian cell culture bioreactors used for large-scale commercial production of these complex proteins (which often require mammalian cells for correct folding and glycosylation).
PART 3: CHALLENGES AND FORMULATION STRATEGIES
Section 6: Handling Peptides/Proteins – In Vitro & In Vivo Challenges (Page 10)
A. Challenges In Vitro (during manufacturing, storage, handling):
Large, Unstable Molecules: Their complex 3D structure is held together by weak non-covalent forces (hydrogen bonds, hydrophobic interactions, ionic bonds). These are easily disrupted.
Susceptibility to Degradation:
Physical Instability: Denaturation (unfolding), aggregation (clumping), adsorption to surfaces (containers, tubing).
Chemical Instability: Hydrolysis (breaking of peptide bonds), deamidation, oxidation (especially Met, Cys, Trp residues), disulfide exchange.
Sensitivity to Environmental Factors: Temperature, pH, agitation, light, and presence of heavy metals can all cause rapid degradation.
Production Difficulty: Historically hard to obtain in large, pure quantities, though recombinant DNA technology has revolutionised this.
B. Challenges In Vivo (after administration):
Immunogenicity: The body's immune system may recognise the therapeutic protein as foreign, generating B-cell (antibodies) and T-cell responses. This can neutralise the drug's effect, cause allergic reactions (including anaphylaxis), or, in rare cases, cross-react with an endogenous protein (e.g., pure red cell aplasia with some erythropoietin formulations).
Proteolytic Degradation: Peptides/proteins are rapidly broken down by a vast array of endopeptidases (cleave internally) and exopeptidases (cleave from ends) present throughout the body, especially in the GI tract, blood, and liver.
Rapid Renal Clearance: The kidneys act as a filter. Small proteins with a molecular weight <30,000 Daltons (30 kDa) are small enough to pass through the glomerular filtration barrier and are quickly excreted in urine, resulting in a very short plasma half-life.
Adsorption/Insolubility: At physiological pH and salt concentrations, proteins can become insoluble and precipitate, or can adsorb non-specifically to tissues or medical devices, leading to unpredictable pharmacokinetics and loss of active drug.
Section 7: Key Formulation Strategies to Overcome Challenges (Page 11)
To address these inherent instabilities, several formulation and modification strategies are employed:
Protein Sequence Modification (Site-Directed Mutagenesis): Genetically engineering the protein to replace susceptible amino acids with more stable ones.
PEGylation: Covalent attachment of polyethylene glycol (PEG) chains to the protein.
Micro/Nanoparticle Encapsulation: Entrapping the protein within a protective polymer or lipid particle.
Permeation Enhancers: Co-administering agents that temporarily and reversibly disrupt epithelial barriers to improve absorption, especially for non-parenteral routes (e.g., surfactants, bile salts, fatty acids).
Enzyme Inhibitors: Co-administering agents that inhibit proteolytic enzymes, protecting the peptide from degradation (e.g., soybean trypsin inhibitor, which inhibits trypsin that cleaves at arginine and lysine residues).
Section 8: Protein Sequence Modification (Site-Directed Mutagenesis) – Page 12
Principle: Using recombinant DNA technology to alter the gene encoding the protein, resulting in a modified protein with one or more amino acid substitutions.
Applications & Benefits:
Enhancing Chemical Stability: For example, replacing an oxidation-prone methionine (Met) with a more stable leucine (Leu) can significantly reduce oxidative degradation.
Improving Physical Stability/Thermal Stability (Tm): Strategically introducing cysteine (Cys) residues can allow the formation of new disulfide bridges (S-S bonds) within the protein. These covalent cross-links reinforce the folded structure, increasing its melting temperature (Tm) – the temperature at which it denatures – making it more resistant to heat and other stresses.
"Protein Engineering" for Other Properties: Altering the protein's size, shape, surface charge, or glycosylation sites can modulate its activity, receptor binding, immunogenicity, or pharmacokinetics.
Section 9: PEGylation (Pages 13-14)
The Process: The covalent attachment of one or more chains of polyethylene glycol (PEG) to the target protein or peptide.
What is PEG? (Page 14):
A synthetic polymer composed of repeating ethylene oxide units
(-O-CH₂-CH₂-)n.It is non-toxic, hydrophilic (water-loving), and neutral (uncharged).
It is highly flexible and extensively hydrated in aqueous solutions.
Mechanism of Action & Benefits (Page 14):
Increased Circulatory Half-Life (5 to 100-fold): The large, hydrated PEG chain creates a "cloud" around the protein. This sterically hinders interactions with:
Proteolytic enzymes (increases protease resistance).
Immune cells and antibodies (decreases immunogenicity).
The glomerular filtration apparatus in the kidneys (reduces renal clearance).
Increased Solubility & Stability: The hydrophilic PEG enhances the protein's solubility in aqueous environments and can help shield it from aggregation and surface adsorption.
Reduced Depot Loss: For subcutaneous or intramuscular injections, PEGylation can reduce the amount of protein lost by being "trapped" at the injection site, improving bioavailability.
Image Description (Page 13): Likely shows a molecular model or schematic diagram of a protein (e.g., a sphere) with long, flexible, wavy polymer chains (PEG) attached at specific sites (e.g., lysine residues or N-terminus). The PEG "cloud" is shown physically shielding the protein from its environment.
Section 10: Encapsulation into Particulate Systems (Pages 15-18)
10.1. General Principle (Page 16):
Concept: Entrapping the peptide/protein drug within a particle made of another material (polymer or lipid).
Purpose:
Protection: The particle's matrix or membrane physically shields the delicate protein from harsh environments (e.g., gastric acid, proteolytic enzymes).
Sustained Release: The rate of drug diffusion out of the particle, or the degradation of the particle matrix, can be controlled to provide prolonged release over hours, days, or even weeks.
Targeting: Particles can be coated with targeting ligands to direct them to specific cells or tissues.
10.2. Types of Materials for Encapsulation (Page 16):
Non-Biodegradable: Remain intact in the body and need to be removed surgically if implanted. Used in long-term implants. Examples: Ethylene co-vinyl acetate, Polymethacrylic acid.
Biodegradable: Break down over time into non-toxic, metabolisable products. Ideal for injectable or implantable systems that don't require removal.
Polymer-based: Poly(lactic-co-glycolic acid) (PLGA) – the most widely used biodegradable polymer. Degrades by hydrolysis into lactic and glycolic acid.
Lipid-based: Phospholipids (e.g., Phosphatidyl choline) – form liposomes, which are vesicles with an aqueous core that can encapsulate hydrophilic proteins.
Image Description (Page 15): Likely shows scanning electron microscopy (SEM) images of insulin-loaded microparticles, highlighting their size and morphology. The caption references a conference presentation on optimising oral insulin formulations.
10.3. Smart/Stimuli-Responsive Particles – pH-Sensitive System (Page 17):
System: A microparticle composed of a poly(methacrylic acid)-PEG (PMA-PEG) copolymer.
Mechanism: This polymer is a polyacid. It exhibits pH-dependent swelling/deswelling called complexation.
At Stomach pH (~2): The carboxylic acid groups on PMA are protonated (-COOH). The polymer is neutral, hydrophobic, and collapsed. The pores in the microparticle are closed, preventing protein release.
At Small Intestine pH (~6.8-7.4): The carboxylic acids deprotonate (-COO⁻), becoming negatively charged. The polymer becomes hydrophilic, and the chains repel each other, causing the matrix to swell. The pores open, allowing the encapsulated protein to release.
Application: This is an ideal system for oral delivery of peptides/proteins. It protects the drug in the stomach and releases it at the primary absorption site (the small intestine).
10.4. Effect of Polymer Type on Release Profile (Page 18):
The choice of polymer dramatically influences the release kinetics and protein stability.
Hydrophilic Polymers (e.g., Gelatin): Water-soluble. They tend to swell rapidly and dissolve/erode quickly, leading to a burst release of the drug. Less suitable for long-term sustained release.
Hydrophobic Polymers (e.g., PLGA): Water-insoluble. Drug release is controlled by slow diffusion through the polymer matrix and gradual hydrolysis of the polymer. Excellent for sustained release. However, the acidic microenvironment created during PLGA degradation and the exposure to organic solvents during manufacturing can denature proteins.
Hybrid Amphiphilic Block Copolymers (e.g., Poly(methyl methacrylate)-PEG, PMMA-PEG): The combination of a hydrophobic block (PMMA) and a hydrophilic block (PEG) can offer advantages.
Good sustained release from the hydrophobic PMMA domains.
Protein structure is better retained because the hydrophobic/hydrophilic balance can be tuned to create a more "protein-friendly" environment during encapsulation and release.
Image Description (Page 18): Likely shows the chemical structure of a PMMA-PEG block copolymer, with x and y representing the number of repeating units in each block, which can be adjusted to tune polymer properties. A graph might compare release profiles (e.g., % release vs. time) for gelatin (rapid burst), PLGA (slow sigmoidal), and a hybrid (intermediate, smooth).
Section 11: Stabilising Excipients (Page 19)
Excipients are added to formulations for specific stabilising effects:
Salts/Ions:
Role: Promote specific, non-covalent interactions that stabilise the native conformation or promote the formation of multimers (e.g., hexamers).
Example: Zinc ions (Zn²⁺) are used in many insulin formulations (e.g., Lente, Ultralente) to promote the formation of stable hexamers, which prolongs its action after subcutaneous injection.
Polyols (e.g., Glycerol, PEG):
Role: Act as co-solvents or stabilisers. They can preferentially hydrate the protein, making it more stable and soluble.
Sugars / Dextran:
Role: In liquid formulations, they can stabilise by displacing water and reducing molecular mobility. In lyophilised (freeze-dried) formulations, they act as lyoprotectants, forming a glassy matrix that protects the protein structure during freezing and drying.
Surfactants (e.g., Polysorbate 80):
Role: Reduce adsorption of the protein to surfaces (e.g., vial walls, syringe barrels) and minimise aggregation caused by interfacial stress (e.g., from shaking or air bubbles). They work by competitively coating hydrophobic surfaces that the protein would otherwise stick to.
PART 4: ADVANCED DELIVERY PLATFORMS AND ROUTES
Section 12: Peptide Micelles (Page 20)
Structure: Self-assembled nanoparticles formed from amphiphilic peptide-based copolymers. They are analogous to lipid micelles.
Composition: Typically consist of:
Hydrophobic Core: Formed from the hydrophobic peptide segments or conjugated hydrophobic polymers. This core can solubilise and protect the therapeutic peptide/protein.
Hydrophilic Shell: Often composed of a PEG corona, which provides steric stabilisation, reduces protein adsorption (stealth effect), and prolongs circulation.
Key Features:
Small Size: 10-50 nm in diameter, similar in scale to viruses. This small size can facilitate tissue penetration.
Controlled Release: Release can be triggered by pH changes (e.g., in endosomes) or redox potential (e.g., high glutathione in cytoplasm) by designing micelles that disassemble under those conditions.
Targeting: The surface of the micelle can be decorated with targeting ligands (e.g., antibodies, folic acid) for active targeting to specific cells.
Image Description (Page 20): A schematic diagram of a peptide micelle, showing a core-shell structure. The core is labelled as containing the drug (peptide/protein), and the shell is labelled as PEG with potential targeting ligands attached. An arrow indicates drug release triggered by an internal stimulus (pH/redox).
Section 13: Routes of Delivery – Overview (Page 21)
Parenteral: By far the most common route (see next section).
Oral / Nasal: Highly desirable but extremely challenging due to barriers (GI degradation, poor permeability, nasal mucociliary clearance).
Transdermal: Delivery through the skin. Challenging due to the skin's impermeability to large molecules, but technologies like microneedle patches are advancing.
Other Routes: Pulmonary (inhalation), Rectal/Vaginal, Ocular – each with specific anatomical barriers and opportunities.
Section 14: Parenteral Delivery – The Dominant Route (Page 22)
Prevalence: Accounts for >95% of protein and peptide therapeutics.
Advantages:
Rapid & Complete Absorption: Directly enters the bloodstream (IV) or is absorbed quickly from the injection site (SC/IM).
Lower Dose Requirement: Avoids degradation and first-pass metabolism, so less drug is needed.
Predictable Pharmacokinetics: More controlled exposure.
Disadvantages:
Safety: Risk of overdosing (if wrong dose given), necrosis (tissue death at injection site).
Local Reactions: Pain, swelling, redness, hypersensitivity reactions.
Patient Compliance: Inconvenience, needle phobia, need for healthcare professional administration or self-injection training, leading to poor adherence for chronic conditions.
Section 15: Oral Delivery Technology – Peptelligence™ (Page 23)
The Challenge: Oral delivery is the "holy grail" for peptides due to ease and compliance, but faces three major barriers in the GI tract:
Acidic Degradation in Stomach.
Proteolytic Enzyme Attack throughout the gut.
Poor Permeability across the intestinal epithelium.
Peptelligence™ Technology (Unigene): A proprietary platform designed to overcome these barriers. Its multi-pronged approach likely includes:
Enteric Coating: Protects the peptide from stomach acid.
Protease Inhibitors: Co-formulated to temporarily inhibit enzymes (e.g., trypsin, chymotrypsin) that would degrade the peptide.
Absorption Enhancers: Agents that temporarily and reversibly open tight junctions between epithelial cells or increase membrane fluidity, allowing the peptide to pass through into the bloodstream.
Image Description (Page 23): Likely a diagram of a Peptelligence™ tablet or capsule, showing an enteric coating and a core containing the peptide drug along with key functional excipients (protease inhibitors, absorption enhancers).
Section 16: Transdermal Delivery – Microneedle Patches (Pages 24-25)
The Challenge: The stratum corneum (outermost layer of skin) is an excellent barrier that prevents the passage of large, hydrophilic molecules like peptides.
Microneedle Technology: Overcomes this by physically bypassing the stratum corneum.
Design: A patch array containing dozens to hundreds of microscopic needles, typically 100-1000 µm in length.
Mechanism: The patch is applied to the skin. The microneedles painlessly pierce the stratum corneum and create transient microscopic channels into the viable epidermis/dermis.
Delivery: The peptide/protein drug, either coated on the needles or embedded in the patch matrix, can then diffuse into the capillaries and enter the systemic circulation.
Advantages:
Minimally Invasive: Virtually painless.
Improved Compliance: Simple application, avoids needles and sharps waste.
Bypasses First-Pass Metabolism.
MacroFlux® (Page 25): An example of a transdermal patch technology platform. It likely uses microneedles or an alternative permeation enhancement technique (e.g., iontophoresis) to facilitate peptide delivery.
Image Description (Page 25): Likely shows a schematic or photograph of a MacroFlux® transdermal patch. It may illustrate the patch structure with its adhesive backing and the array of microneedles, or a diagram showing the microneedles penetrating the stratum corneum and releasing the drug into the dermal microcirculation.
Section 17: Summary and Future Outlook (Page 26)
Growth Sector: Protein/peptide pharmaceuticals are the most rapidly growing sector in the pharmaceutical industry, driven by biotechnology advances and the ability to target diseases with high specificity.
Broad Therapeutic Potential: They are at the forefront of developing treatments for complex diseases, including Alzheimer's, cancer, multiple sclerosis, auto-immune diseases, and many more.
Delivery is the Key Challenge: While the parenteral route dominates today, significant research and commercialisation efforts are focused on developing extravascular routes (oral, transdermal, pulmonary) to improve patient compliance and quality of life.
Commercial Success: Newer formulation and delivery platforms (e.g., PEGylated proteins, liposomal formulations, oral peptide technologies, microneedle patches) are not just academic concepts; they are being successfully commercialised, demonstrating the viability of these advanced approaches