Pharmacy 361

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Pharmacy 361

• Protein Formulation Delivery
• David Wishart
• david.wishart_at_ualberta.ca
• 3-41 Athabasca

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Todays lecture notes are available at

• http//redpoll.pharmacy.ualberta.ca

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The Human Genome Project

• First Draft completed on June 26, 2000
• 3,260,000,000 bp on 24 chromosomes
• 3,201,762,515 bases sequenced (98)
• 23,531 - 31,609 genes (predicted)
• All FDA approved drugs target just 417 different
  proteins

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Proteins

• Polypeptides composed of covalently linked amino
  acids
• Polypeptides with lt40 amino acids are called
  peptides
• Polypeptides with gt40 amino acids are called
  proteins
• Function of a protein determined by its
  non-covalent 3D structure

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Amino Acids
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Polypeptides
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Protein Structure
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Protein Pharmaceuticals

• gt600 FDA approved protein drugs
  (http//208.69.231.70/biopharma/results7.lasso)
• gt30 are recombinant (rDNA) proteins
• Protein pharmaceutical sales currently approach
  46 billion/yr
• By 2011 they are expected to reach 52 billion/yr

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Finding More About Protein Drugs
http//www.drugbank.ca or just google drugbank
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DrugBank DrugCards
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Searching DrugBank
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Searching DrugBank
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Searching By Text
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DrugBank Statistics
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Part-time Jobs Available

• Up to 25/hour
• Work from home, flexible hours
• Learn more about drugs, drug actions,
  pharmacology
• Annotation of DrugBank2
• Contact Alex Frolkis at 492-5009 (between 9 am
  and 5 pm)

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Classes of Protein Pharmacueticals

• Vaccines (peptides, parts of proteins, killed
  bacteria)
• Peptides (oxytocin, pitocin)
• Blood products (Factor X, Factor VIII, gamma
  globulin, serum albumin)
• Recombinant therapeutic proteins (herceptin,
  humulin, alferon, etc.)

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Vaccines

• Diptheria (Corynebacterium diphtheriae) -
  diptheria toxin
• Tetanus (Clostridium tetani) - tetanus toxin
• Whooping cough (Bordetella pertussis) - acullelar
  extract

Tetanus Toxin HC Fragment
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Therapeutic Proteins

• Insulin (diabetes)
• Interferon b (relapsing MS)
• Interferon g (granulomatous)
• TPA (heart attack)

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Therapeutic Proteins

• Actimmune (If g)
• Activase (TPA)
• BeneFix (F IX)
• Betaseron (If b)
• Humulin
• Novolin
• Pegademase (AD)

• Epogen
• Regranex (PDGF)
• Novoseven (F VIIa)
• Intron-A
• Neupogen
• Pulmozyme
• Infergen

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The Problem with Proteins

• Very large and unstable molecules
• Structure is held together by weak noncovalent
  forces
• Easily destroyed by relatively mild storage
  conditions
• Easily destroyed/eliminated by the body
• Hard to obtain in large quantities

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The Problem with Proteins(in vivo - in the body)

• Elimination by B and T cells
• Proteolysis by endo/exo peptidases
• Small proteins (lt30 kD) filtered out by the
  kidneys very quickly
• Unwanted allergic reactions may develop (even
  toxicity)
• Loss due to insolubility/adsorption

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The Problem with Proteins(in vitro - in the
bottle)
Noncovalent Covalent

• Denaturation
• Aggregation
• Precipitation
• Adsorption

• Deamidation
• Oxidation
• Disulfide exchange
• Proteolysis

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Noncovalent Processes
Denaturation Adsorption
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Noncovalent Processes
Aggregation Precipitation
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Covalent Processes

• Deamidation - conversion of Asn-Gly sequences to
  a-Asp-Gly or b-Asp-Gly
• Oxidation - conversion RSR to RSOR, RSO2R or
  RSO3R (Met Cys)
• Disulfide exchange - RS- RS-SR goes to
  RS-SR RS- (Cys)
• Proteolysis - Asp-Pro, Trypsin (at Lys) or
  Chymotrypsin (at Phe/Tyr)

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Deamidation
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How to Deal with These Problems?
Storage
Formulation
Delivery
Pharmaceutics
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Storage - Refrigeration

• Low temperature reduces microbial growth and
  metabolism
• Low temperature reduces thermal or spontaneous
  denaturation
• Low temperature reduces adsorption
• Freezing is best for long-term storage
• Freeze/Thaw can denature proteins

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Storage - Packaging

• Smooth glass walls best to reduce adsorption or
  precipitation
• Avoid polystyrene or containers with silanyl or
  plasticizer coatings
• Dark, opaque walls reduce hn oxidation
• Air-tight containers or argon atmosphere reduces
  air oxidation

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Storage - Additives

• Addition of stabilizing salts or ions (Zn for
  insulin)
• Addition of polyols (glycerol and/or polyethylene
  glycol) to solubilize
• Addition of sugars or dextran to displace water
  or reduce microbe growth
• Use of surfactants (CHAPS) to reduce adsorption
  and aggregation

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Storage - Freeze Drying

• Only cost-effective means to prepare solid,
  chemically active protein
• Best for long term storage
• Removes a considerable amount of water from
  protein lattice, so much so, that some proteins
  are actually deactivated

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Freeze Drying

• Freeze liquid sample in container
• Place under strong vacuum
• Solvent sublimates leaving only solid or
  nonvolatile compounds
• Reduces moisture content to lt0.1

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Sublimation vs. Melting
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Protein Pharmaceutics
Storage
Formulation
Delivery
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The Problem with Proteins(in vivo)

• Elimination by B and T cells
• Proteolysis by endo/exo peptidases
• Small proteins (lt30 kD) filtered out by the
  kidneys very quickly
• Unwanted allergic reactions may develop (even
  toxicity)
• Loss due to insolubility/adsorption

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Protein Formulation

• Protein sequence modification (site directed
  mutagenisis)
• PEGylation
• Proteinylation
• Microsphere/Nanosphere encapsulation
• Formulating with permeabilizers

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Site Directed Mutagenesis
E343H
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Site Directed Mutagenesis

• Allows amino acid substitutions at specific sites
  in a protein
• i.e. substituting a Met to a Leu will reduce
  likelihood of oxidation
• Strategic placement of cysteines to produce
  disulfides to increase Tm
• Protein engineering (size, shape, etc.)

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PEGylation

CH-CH-CH-CH-CH-CH-CH-CH-CH-CH
OH OH OH
OH OH OH OH OH OH OH
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PEGylation

• PEG is a non-toxic, hydrophilic, FDA approved,
  uncharged polymer
• Increases in vivo half life (4-400X)
• Decreases immunogenicity
• Increases protease resistance
• Increases solubility stability
• Reduces depot loss at injection sites

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Proteinylation

Protein Drug ScFv (antibody)
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Proteinylation

• Attachment of additional or secondary
  (nonimmunogenic) proteins for in vivo protection
• Increases in vivo half life (10X)
• Cross-linking with Serum Albumin
• Cross-linking or connecting by protein
  engineering with antibody fragments

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Microsphere Encapsulation
100 mm
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Encapsulation

• Process involves encapsulating protein or peptide
  drugs in small porous particles for protection
  from insults and for sustained release
• Two types of microspheres
• nonbiodegradable
• biodegradable

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Types of Microspheres

• Nonbiodegradable
• ceramic particles
• polyethylene co-vinyl acetate
• polymethacrylic acid/PEG
• Biodegradable (preferred)
• gelatin
• polylactic-co-glycolic acid (PLGA)

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PLGA - Structure
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Microsphere Release

• Hydrophilic (i.e. gelatin)
• best for burst release
• Hydrophobic (i.e. PLGA)
• good sustained release (esp. vaccines)
• tends to denature proteins
• Hybrid (amphipathic)
• good sustained release
• keeps proteins native/active

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Release Mechanisms
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Peptide Micelles
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Peptide Micelles

• Small, viral sized (10-50 nm) particles
• Similar to lipid micelles
• Composed of peptide core (hydrophobic part) and
  PEG shell (hydrophilic part)
• Peptide core composition allows peptide/protein
  solubilization
• Also good for small molecules

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Peptide Synthesis
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Peptide-PEG monomers
Hydrophobic block
Hydrophilic block
Peptide
PEG
CH-CH-CH-CH-CH-CH-CH-CH-CH-CH
OH OH OH
OH OH OH OH OH OH OH
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Peptide Micelles
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Targeted Micelles
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Nanoparticles for Vaccine Delivery to Dendritic
Cells

• Dendritic Cells -sentries
• of the body
• Eat pathogens and present
• their antigens to T cells
• Secret cytokines to direct
• immune responses

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Nanoparticles for Vaccine Delivery

• Mimic pathogen surface characteristics
• Antigen for controlled delivery within Dendritic
  Cells
• Selective activation of cytokine genes in
  Dendritic Cells
• Applications in Therapeutic Vaccines (e.g.,
  cancer, AIDS, HBV, HCV)

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Polymeric Nanoparticle Uptake by Human DCs
Confocal Image
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Permeabilizers (Adjuvants)

• Salicylates (aspirin)
• Fatty acids
• Metal chelators (EDTA)
• Anything that is known to punch holes into the
  intestine or lumen

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Protein Formulation (Summary)

• Protein sequence modification (site directed
  mutagenisis)
• PEGylation
• Proteinylation
• Microsphere/Nanosphere encapsulation
• Formulating with permeabilizers

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Protein Pharmaceutics
Storage
Formulation
Delivery
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Routes of Delivery

• Parenteral (injection)
• Oral or nasal delivery
• Patch or transdermal route
• Other routes
• Pulmonary
• Rectal/Vaginal
• Ocular

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Parenteral Delivery

• Intravenous
• Intramuscular
• Subcutaneous
• Intradermal

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Parenteral Delivery

• Route of delivery for 95 of proteins
• Allows rapid and complete absorption
• Allows smaller dose size (less waste)
• Avoids first pass metabolism
• Avoids protein unfriendly zones
• Problems with overdosing, necrosis
• Local tissue reactions/hypersensitivity
• Everyone hates getting a needle

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Exubera (Inhaled Insulin)

• Exubera, a dry-powder form of insulin, is inhaled
  with a special device similar to an asthma
  inhaler
• Exubera normalized blood sugar levels as well as
  injections did
• Patients taking inhaled insulin also reported
  greater satisfaction and quality of life (for 18
  only)
• About 1/5 study subjects developed a mild cough
  with inhaled insulin
• Product pulled in Oct. 2007

Pfizer
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Oral Insulin (Oralin)
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Oral Insulin (Oralin/Oral-lyn)

• Bucchal aerosol delivery system developed by
  Generex (Approved in Ecaudor and India)
• Insulin is absorbed through thin tissue layers in
  mouth and throat
• Insulin is formulated with a variety of additives
  and stabilizers to prevent denaturation on
  aerosolization and to stabilize aerosol particles

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BioSantes BioOral Insulin

• The BioOral formulation was developed by
  aggregating caseins (the principle protein in
  milk) around a proprietary formulation of CAP
  (calcium phosphate nanoparticle), polyethylene
  glycol (PEG, a polymer) and insulin by scientists
  at BioSante's research center

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Oral Delivery by Microsphere
pH 2 pH 7
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pH Sensitive Microspheres

• Gel/Microsphere system with polymethacrylic acid
  PEG
• In stomach (pH 2) pores in the polymer shrink and
  prevent protein release
• In neutral pH (found in small intestine) the
  pores swell and release protein
• Process of shrinking and swelling is called
  complexation (smart materials)

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Patch Delivery
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Mucoadhesive Patch

• Adheres to specific region of GI tract
• Ethylcellulose film protects drugs from
  proteolytic degradation
• Composed of 4 layers
• Ethylcellulose backing
• Drug container (cellulose, citric acid)
• Mucoadhesive glue (polyacrylic acid/PEG)
• pH Surface layer (HP-55/Eudragit)

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Patch Delivery
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GI-MAPS Layers

• pH sensitive surface layer determines the
  adhesive site in the GI tract
• Gel-forming mucoadhesive layer adheres to GI
  mucosa and permits controlled release - may also
  contain adjuvants
• Drug containing layer holds powders, dispersions,
  liquids, gels, microspheres,
• Backing layer prevents attack from proteases and
  prevents luminal dispersion

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Transdermal Patches
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Transdermal Patches

• Proteins imbedded in a simple matrix with
  appropriate additives
• Patch is coated with small needles that penetrate
  the dermal layer
• Proteins diffuse directly into the blood stream
  via capillaries
• Less painful form of parenteral drug delivery

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Close-up of Patch Pins
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MacroFlux Transdermal Patch
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The Future

• Greater use of Nanotechnology in biopharmaceutics
  (nanopharm)
• Using cells as Protein Factories or as
  targetable Nanosensors Nanorobots
• Artificial or Synthetic Cells as drug delivery
  agents

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Smart Pills
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Smart Pills (Nano-Robots)
Unlikely Likely
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Micromachined Biocapsules
Artificial Islet Cells - Tejal Desai (UI)
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Micromachining

• Uses photolithography or electron beam etching to
  carved small (5 nm) holes into metal (titanium)
  plates
• Porous plates are placed over small metal boxes
  containing islet cells
• Insulin (2 nm) leaks out through diffiusion, but
  antibodies are too big (10 nm) to get in

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Biocapsules
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Summary

• Protein pharmaceuticals are (and will be) the
  most rapidly growing sector in the pharmaceutical
  repertoire
• Most cures for difficult diseases (Alzheimers,
  cancer, MS, auto-immune diseases, etc.) will
  probably be found through protein drugs

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Summary

• BUT Proteins are difficult to work with
• Most protein delivery is via injection
• Newer methods are appearing
• Oral delivery using smart materials is looking
  promising
• Over the coming 3-4 years many more protein drugs
  will be taken orally