Pharmacy 610

1
Pharmacy 610

• Challenges in Peptide Protein Formulation
  Delivery
• David Wishart
• david.wishart_at_ualberta.ca
• Rm. 2123 DPC

2
Outline (Part I)

• Protein Structure (Review)
• Protein Energetics (without H2O)
• Protein Energetics (with H2O)
• Water Structure
• The Hydrophobic Effect
• Hydrogen Bonding and the Hydrogen Bond Myth

3
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

4
Amino Acids
5
Glycine and Proline
H
C
C
COOH
H2N
COOH
HN
H
H
P
G
6
Aliphatic Amino Acids
CH3
CH3
CH3
CH3
V
I
C
C
COOH
H2N
COOH
H2N
H
H
CH3
CH3
CH3
A
L
C
COOH
H2N
C
COOH
H2N
H
H
7
Aromatic Amino Acids
N
N
N
W
H
C
C
COOH
H2N
COOH
H2N
OH
H
H
Y
F
C
C
COOH
H2N
COOH
H2N
H
H
8
Charged Amino Acids
H
-
COO

N
NH3
D
R
C
NH
COOH
H2N

C
H
NH3
COOH
H2N
-
COO
H
E
K
C
C
COOH
H2N
COOH
H2N
H
H
9
Polar Amino Acids
OH
CH3
CONH2
N
T
C
C
COOH
H2N
COOH
H2N
H
H
CONH2
OH
Q
S
C
C
COOH
H2N
COOH
H2N
H
H
10
Sulfo-Amino Acids
CH3
S
SH
C
C
COOH
H2N
COOH
H2N
H
H
M
C
11
Polypeptides
12
Levels of Protein Structure
13
Protein Structure
14
Protein Energetics (without water)
15
Newtonian Approximation

• Proteins can be treated as hard spheres with
  spring-like bonds and partial charges

16
Standard Energy Function
E
Kr(ri - rj)2 Kq(qi - qj)2 Kf(1-cos(nfj))2
qiqj/4perij Aij/r6 - Bij/r12 Cij/r10 -
Dij/r12
Bond length Bond bending Bond torsion Coulomb van
der Waals H-bond
17
Energy Terms
r
f
q
Kr(ri - rj)2
Kq(qi - qj)2
Kf(1-cos(nfj))2
Stretching Bending
Torsional
18
Energy Terms
r
r
r
qiqj/4perij
Aij/r6 - Bij/r12
Cij/r10 - Dij/r12
Coulomb van der Waals H-bond
19
Standard Energy Function
E
Kr(ri - rj)2 Kq(qi - qj)2 Kf(1-cos(nfj))2
qiqj/4perij Aij/r6 - Bij/r12 Cij/r10 -
Dij/r12
Kr 600 kcal/Mol/A2 Kq 60 kcal/Mol/rad2 Kf 1
kcal/Mol/rad2 5 kcal/Mol 0.1 kcal/Mol 1-3
kcal/Mol
20
An Energy Surface
High Energy
Low Energy
Overhead View Side View
21
A More Realistic Protein Energy Surface
The Folding Funnel
22
Standard Energy Functions

• Standard energy functions are incapable of
  explaining many of the key stabilizing features
  or proteins
• Furthermore, these functions are incapable of
  distinguishing completely incorrect protein folds
  from completely correct folds
• SOMETHING IS MISSING..

23
Protein Energetics (with water)
24
Water
25
Water Structure

• Water has four near- tetrahedrally arranged,
  sp3-hybridized electron pairs
• Two are associated with the hydrogen atoms
  (positive charges)
• Two are lone pairs from the oxygen atom (negative
  charges)

26
Water Structure

• Liquid water will form transient, short-lived
  supramolecular structures or hydrogen bond
  networks
• Small clusters of four water molecules may come
  together to form what are called bicyclo-octamers

27
Water Structure

• These bicyclo-octamers may cluster with each
  other to form highly symmetric 280-molecule
  icosahedral water clusters that are able to
  interlink and tessellate throughout space

28
Water Structure is Responsible For...

• Unusually high boiling point (100 oC vs. -10 oC
  for NH3)
• Unusually high melting point for ice (0 oC vs
  -160 oC for liquid O2)
• a density maximum while liquid

• High surface tension (73 erg/cm2 vs 25 erg/cm2
  for EtOH)
• Negative volume of melting (ice shrinks on
  melting)
• High dielectric constant (78.5)

29
Water Structure

• Can be perturbed by the presence of anions or
  cations which will form hydration shells and
  alter the preferred network of H bonds
• Can be perturbed by the presence of hydrophobic
  organic molecules which create clathrate cages
  or cavities in water (i.e. break H bonds)

30
The Hoffmeister Series

• Developed by Hoffmeister in 1888 from the ranking
  of various ions toward their ability to
  precipitate a mixture of hen egg white proteins

31
Ions in Water

• Ions break up the natural hydrogen bonding
  structure of H2O (equivalent to heating or
  pressurizing)
• Net effect is reduced viscosity
• Chaotropes or denaturants disrupt H2O structure
  (weak H2O interactions)
• Kosmotropes or stabilizers enhance H2O structure
  (strong H2O interactions)

32
Sulfate Hydration Shell
Kosmotrope
Symmetrical dodecahedral arrangement of 16 water
molecules
33
Protein Stability

• Optimum stabilization of peptides and proteins by
  salt requires a mixture of a kosmotropic anion
  with a chaotropic cation.

34
Water Structure

• Can be perturbed by the presence of anions or
  cations which will form hydration shells and
  alter the preferred network of H bonds
• Can be perturbed by the presence of hydrophobic
  organic molecules which create clathrate cages
  or cavities in water (i.e. break H bonds)

35
Clathrate Cages
Choline Fragments in Water Cages
36
Outline (Part I)

• Protein Structure (Review)
• Protein Energetics (without H2O)
• Protein Energetics (with H2O)
• Water Structure
• The Hydrophobic Effect
• Hydrogen Bonding and the Hydrogen Bond Myth

37
The Hydrophobic Effect

• Phenomenological effect describing the
  insolubility of non-polar atoms and molecules in
  water (separation of oil and water)
• Arises from the energetic cost of disrupting
  water structure (breaking hydrogen bonds to form
  clathrates)
• Often called an entropic effect

38
The Hydrophobic Effect
Water Cavity Formation
Filling With Solute
DG -TDS DH
39
Cavity Formation
DGdis -TDS 15cal/mol A2
40
Cavity Formation

• Water is an elastic solvent or liquid
• Energy of forming a cavity is equal to the
  surface tension (g) of the solvent multiplied by
  the change in surface area (DA) to accommodate
  the solute

DG gDA
41
Cavity Formation

• The cost of creating cavities in water is the
  reason that round oil droplets form when
  oil/vinegar dressing is shaken -- spheres allow
  the maximum volume to be housed in the minimum
  surface area
• This explains why proteins are essentially
  densely packed spheres

42
Protein Packing
Loose Packing Dense Packing Protein
Proteins are Densely Packed
43
Radius Radius of Gyration

• RAD 3.875 x NUMRES 0.333 (Folded)
• RADG 0.41 x (110 x NUMRES) 0.5 (Unfolded)

Radius Radius of Gyration
44
Effective Surface Tension

• While the surface tension in water is supposed to
  be constant (at constant T, P or salt
  concentration) we can speak of effective
  surface tension for specific types of solutes or
  atoms

g(C/S) 16 cal/mol Å2
g(N) -50 cal/mol Å2
g(N/O) -6 cal/mol Å2
g(O-) -24 cal/mol Å2
45
The Hydrophobic Effect and Surface Tension

• This microscopic surface tension approach allows
  one to calculate the Free Energy of dissolving a
  complex molecule (composed of polar and nonpolar
  atoms) into water
• It can also be used to calculate the
  stabilization energy of proteins (at room
  temperature)

46
Protein Polar and Nonpolar Surface Area
Nonpolar (C/S) Polar (N or N) Polar (O or O-)
47
Accessible Surface Area
Reentrant Surface
Accessible Surface
Solvent Probe
Van der Waals Surface
48
Accessible Surface Area
Reentrant Surface
Accessible Surface
Solvent Probe
Van der Waals Surface
49
Accessible Surface Area (ASA)

• If proteins were perfect spheres then ASA V0.66
• Approximate ASA ASA 7.11 x MW
  0.718
• ASA of unfolded protein ASA(U) S AAi x ASAi
• Buried Surface Area BASA ASA(U) - ASA

50
Buried Surface Area (BASA) Fractional Burial
(FB)

• For an average protein
• ASA (NP) 0.35 x BASA
• ASA (P) 0.61 x BASA
• ASA (Q) 0.04 x BASA
• BASA can be estimated from a proteins amino acid
  composition BASA S AAi x FBi

51
Free Energy of Folding

• Using these values or measurements derived from
  programs to calculate surface areas of molecules
  it is possible to calculate the energy required
  to fold (or unfold) a protein

Unfolded Folded
DG(N) -2440 kcal DG(C) 3245 kcal DG(O)
-1245 kcal
DG(N) -3245 kcal DG(C) 5280 kcal DG(O)
-2455 kcal
DG -20 kcal/M
52
The Hydrophobic Effect

• Unlike the situation seen when protein structures
  and energetics were modelled without water we can
  see it is possible to roughly calculate protein
  stabilization energies if water is properly
  accounted for
• The hydrophobic effect is often sufficient to
  distinguish incorrect structures from correct
  structures

53
Protein Energetics

• When solvent effects are included we can see the
  the energy of stabilization for proteins is
  essentially a small difference between many large
  terms
• A small error in any one of these large terms
  will lead to erroneous predictions -- this is
  what makes it so difficult to calculate
  energies for proteins and protein complexes

54
Hydrophobic Effect and Temperature

• The free energy on going from the native (N)
  state to the denatured (D) state is given by
• Polar and nonpolar molecules respond differently
  to temperature changes

55
Thermal Effects

• Proteins can be denatured by either heat or by
  cold
• Most proteins are denatured at temperatures gt 50
  oC
• Some proteins are denatured at temperatures lt 5
  oC
• The optimum temperature for protein stability is
  20-40 oC

56
Electrostatic Effects


-



-
-


-
Water Cavity Formation
Filling With Solute
DG -TDS DH
57
Born Free Energy
e2
radius a charge q
e1
e1
DG q2(1/e1 - 1/e2) ?40Z2/a kcal/mol
2a
58
Born Free Energy

• The burial of charged or partially charged
  molecules in a low dielectric region (interior of
  a micelle, inside a protein) is energetically
  unfavorable
• The movement of a charged molecule from a low
  dielectric region to a high dielectric region
  (surface of a protein) is energetically favorable

59
What About H-bonds?
60

61

Hydrogen Bonds In Proteins Form Dipoles
62
Onsager (dipole) Free Energy
e2
radius a charge q dipole dis r
e1
e1
DG q2r (2e2 - 2e1)
(2e2 e1)
e1a3
63
Onsager (dipole) Free Energy

• Substitution of standard dielectric values for
  water (e2 80) and for the interior of a protein
  (e1 4) yields a positive free energy
• Formation of hydrogen bonds inside proteins in
  unfavorable!!!
• Hydrogen bonds are a consequence of protein
  folding -- not a cause

64
Folded Proteins...

• Maximize number of hydrogen bonds
• Maximize buried nonpolar ASA
• Maximize exposed polar ASA
• Minimize interstitial cavities or spaces

65
Folded Proteins...

• Minimize number of bad contacts
• Minimize number of buried charges
• Minimize radius of gyration
• Minimize covalent and noncovalent (van der Waals
  and coulombic) energies

66
Conclusions (Part I)

• Proteins are unusually large molecules held
  together by weak non-covalent forces
• The most important of these forces is the
  hydrophobic effect
• The delicate balance between strong opposing
  forces means that proteins can be difficult to
  handle and formulate

67
Outline (Part II)

• Protein Pharmaceuticals
• The Problem with Proteins
• Protein Drug Storage
• Protein Drug Formulation
• Protein Drug Delivery
• Novel and Emerging Methods

68
Protein Pharmaceuticals

• gt100 FDA approved protein drugs
  (http//www.bio.org/aboutbio/drugs.html)
• gt80 are recombinant proteins
• Protein pharmaceutical sales currently approach
  35 billion/yr
• By 2004 they are expected to reach 43 billion/yr

69
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.)

70
Vaccines

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

Tetanus Toxin HC Fragment
71
Protein Pharmaceuticals

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

72
Protein Pharmaceuticals

• 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

73
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

74
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

75
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The Problem with Proteins(in vitro - in the
bottle)
Noncovalent Covalent

• Denaturation
• Aggregation
• Precipitation
• Adsorption

• Deamidation
• Oxidation
• Disulfide exchange
• Proteolysis

77
Noncovalent Processes
Denaturation Adsorption
78
Noncovalent Processes
Aggregation Precipitation
79
Noncovalent Processes

• Almost all non-covalent processes or protein
  pharmaceutical inactivation arise because of
  hydrophobic interactions -- either between the
  protein and the bottle or between the protein
  and other proteins
• Recall the hydrophobic effect and the Hoffmeister
  series

80
Monitoring Denaturation and/or Aggregation
81
Monitoring Denaturation and/or Aggregation
82
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)

83
Deamidation
84
Proteolysis
Serine Protease Catalytic Triad
85
Monitoring Covalent Modifications In Proteins
86
Monitoring Covalent Modifications In Proteins
87
How to Deal with These Problems?
Storage
Formulation
Delivery
Pharmaceutics
88
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

89
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

90
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

91
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

92
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

93
Sublimation vs. Melting
94
Protein Pharmaceutics
Storage
Formulation
Delivery
95
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

96
Protein Formulation

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

97
Site Directed Mutagenesis
E343H
98
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.)

99
PEGylation

CH-CH-CH-CH-CH-CH-CH-CH-CH-CH
OH OH OH
OH OH OH OH OH OH OH
100
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

101
Proteinylation

Protein Drug ScFv (antibody)
102
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

103
Microsphere Encapsulation
100 mm
104
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

105
Types of Microspheres

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

106
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

107
Release Mechanisms
108
Peptide Micelles
109
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

110
Peptide Synthesis
111
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
112
Peptide Micelles
113
Targeted Micelles
114
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

115
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)

116
Polymeric Nanoparticle Uptake by Human DCs
Confocal Image
117
Permeabilizers (Adjuvants)

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

118
Protein Formulation

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

119
Protein Pharmaceutics
Storage
Formulation
Delivery
120
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121
Routes of Delivery

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

122
Parenteral Delivery

• Intravenous
• Intramuscular
• Subcutaneous
• Intradermal

123
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

124
Oral Insulin
125
Oral Insulin

• Bucchal aerosol delivery system developed by
  Generex
• 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

126
Oral Delivery by Microsphere
pH 2 pH 7
127
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)

128
Patch Delivery
129
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)

130
Patch Delivery
131
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

132
Transdermal Patches
133
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

134
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

135
Smat Pills (Nano-Robots)
Unlikely Likely
136
Micromachined Biocapsules
Artificial Islet Cells - Tejal Desai (UI)
137
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

138
Biocapsules
139
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

140
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
• By 2010 many protein drugs will be taken orally

141
Protein Prodrugs
142
Ricin
Ribotoxin (A chain)
Lectin (B chain)
143
Ricin Toxin (A chain)
144
Ricin Activiation

• Ricin is not catalytically active until it is
  proteolytically cleaved. This splits the
  polypeptide into the A chain and the B chain
  still linked by a single disulfide bond. Inside
  the cell this disulfide bond is reduced and leads
  to the generation of a free A chain

145
Ricin Uptake

• (1-3) Ricin taken up by endocytosis via coated
  pits and vesicles
• (4-6) Ricin moved intracellularly and some
  excreted
• 7 Ricin released into cytosol