Download Delivery of Insulin-Like Growth Factor

“Delivery of Insulin-Like Growth
Factor-I to the Rat Brain and Spinal
cord along Olfactory and Trigeminal
Pathways Following Intranasal
Administration.”
Thorne, R. G., Pronk, G. J., Padmanabhan, V., and
Frey II, W. H.
Presented by Mattia Migliore
October 13, 2005
Introduction:




Neurotrophic factors are proteins that help
promote the development, survival, repair, and
proliferation of neurons.
Term derived from the Greek “trophimos,”
which means nourishing or nutritious.
Usually hydrophilic, basic proteins with
molecular weights ranging from 5-30 kDa.
Prototypic neurotrophic factor is nerve growth
factor (NGF), but more than 20 different
neurotrophic factors have been identified.
Introduction (Cont.):


Neurotrophic factors have been shown to exert
their pharmacological effects at concentrations
of only nanomolar to femtomolar ranges.
Potential curative treatments for
neurodegenerative disorders including
Parkinson’s disease, Alzheimer’s disease, and
to reverse the neurological damage caused by
strokes and/or head injuries.
Limitations to the therapeutic use of
neurotrophic factors for the
treatment of neurodegenerative
diseases:
Exogenously administered neurotrophic
factors cannot reach their site of
action because they do not cross the
blood-brain barrier.
Limitations to the therapeutic use of
neurotrophic factors for the
treatment of neurodegenerative
diseases (cont.):


Also, they have very short half-lives due
to their rapid clearance and degradation
by plasma hydrolytic enzymes.
Currently, the only ways to deliver a
neutrophic factor into the brain are by
either injecting the peptide
intraparenchymally, or by administering
the peptide into the cerebroventricular
(IVC) cavity.
Problems Associated with
Intraparenchymal and IVC
Administration:


Surgery to implant the catheter required
for administration is extremely invasive.
Patients at risk for:
1. Infection (including sepsis).
2. Encephalitis.
3. Embolic and/or hemorrhagic
strokes.
4. Seizures.
5. Death.
Why can’t neurotrophic
factors cross the blood-brain
barrier?
Blood-Brain Barrier Permeability:


Presence of tight junctions in the
cerebral vasculature limits what gets in
and out of the brain.
Only lipophilic molecules with
molecular weights of less than 500 Da
are able to cross the blood-brain barrier
(unless transport is receptor mediated).
The Blood-Brain Barrier:
From: http://www.ualberta.ca/~csps/JPPS6(2)/A.Misra/Figure%201.gif
Tight Junctions:
Hawkins and Davis. Neurovascular Unit in Health and Disease. 2005.
Transport of Substances Across
the Blood-Brain Barrier:
Begley et al, 2000.
Neurotrophic Factor Families:
1. Neurotrophin family:
a. NGF.
b. BDNF.
c. Neurotrophin-3 (NT-3).
d. Neurotrophin-4/5 (NT-4/5).
e. NT-6 and NT-7 (only in fish).
Neurotrophic Factor Families
(Cont.):
2. Fibroblast growth factor family (more than
20):
a. aFGF or FGF-1.
b. bFGF or FGF-2.
3. Neurokine family:
a. Ciliary neurotrophic factor
(CNTF).
b. Leukemia inhibitory factor.
c. Cardiotrophin-1(CT-1).
Neurotrophic Factor Families
(Cont.):
4. Transforming growth factor beta family:
a. TGFβ.
b. Bone Morphogenic proteins (BMP).
c. Growth differentiation factors (GDF).
d. Glial cell derived neurotrophic factor
(GDNF).
e. Neurturin (NTN).
f. Artemin (ART).
g. Persephin (PSP).
Neurotrophic Factor Families
(Cont.):
5. Epidermal growth factor family:
a. EGF.
b. Transforming growth factor α.
6. Platelet derived growth factors.
7. Activity-dependent growth factor (ADNF).
8. Insulin-like growth factor family:
a. IGF-1.
b. IGF-2.
Insulin-Like Growth Factors:

Functions:
1.
2.
3.
4.


Cell growth.
Differentiation.
Migration.
Survival.
IGF-I is a single polypeptide chain with a 70
amino acid sequence, and a molecular weight
of 7.65 KDa.
IGF-I has similar 3-D structure to proinsulin.
Insulin-like Growth Factors
(Cont.):




IGF-II is a 67 amino acid peptide with
structural homology to IGF-I.
IGF-I is believed to promote neuronal survival
and/or regeneration, and is a much more potent
growth factor than IGF-2.
IGF-1 is the main mediator of growth
hormone’s actions.
In humans, the IGF-I gene has been mapped to
chromosome 12.
Endocrine Vs. Paracrine
Release.

Endocrine release: IGF-I is primarily
made in the liver in response to growth
hormone stimulation.

IGF-II synthesis, on the other hand, appears to
be independent from the influences of GH.
Paracrine release: IGF-I can also be
synthesized locally in response to various
trophic hormones (seemingly independently
from GH).

Synthesis of Insulin-like growth
factors:
Transport of Insulin like growth
factors:





IGFs circulate in the blood bound to IGF-binding
proteins (IGFBPs).
Functions of IGFBPs are to transport and to store
IGFs.
There are 6 different types of IGFBPs and they all
bind to both IGFs.
IGFBPs regulate the interaction of IGFs with their
receptors.
They are regulated through phosphorylation,
proteolysis, and through association with cell
components.
IGFs receptors:






Found in all tissues.
Members of the tyrosine kinase receptor family.
Consist of 2 alpha and 2 beta subunits joined by
disulfide cross bridges.
Insulin receptors can also bind IGF-1 and IGF2.
IGF-1 receptor can also bind IGF-2 and insulin.
IGF-2 receptor can’t bind insulin, but it can bind
IGF-1.
Hypothesis:
“Intranasally delivered IGF-1 can bypass
the blood-brain barrier via olfactory and
trigeminal associated extracellular
pathways to rapidly elicit biological
effects at multiple sites within the brain
and spinal cord.”
Intranasal Administration:

Intranasal drug delivery takes advantage of
the presence of an incomplete blood brain
barrier in the olfactory epithelium (Graff and
Pollack, 2005).

The olfactory nerves are able to
completely bypass the blood brain barrier,
and drugs that can be taken up by these
neurons can be transported directly into
the brain (Graff and Pollack, 2005).
(www.sfn.org/content/Publications/BrainBriefings/smell.htm)
Intranasal Administration
(cont.):


There are three main mechanisms by
which drugs are absorbed intranasally:
transcellularly, paracellularly, and via
axonal transport (Illum, 2003).
Transcellular drug transport involves the
olfactory epithelium and the process of
endocytosis, either via receptor mediated
uptake, or via passive diffusion (Illum, 2003).
Intranasal Drug Delivery:
Paracellular Drug Absorption
http://www.nastech.com/img/img_junction_structure.jpg.
Intranasal Drug Delivery:
Intraneuronal Route of Drug
Absorption:
www.bionn.com/book/biology/imag/8/8.16
Cranial Nerves:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Olfactory Bulb.
Optic Nerve.
Oculomotor Nerve.
Trochlear Nerve.
Trigeminal Nerve.
Abducens Nerve.
Facial Nerve.
Vestibulocochlear Nerve.
Glossopharyngeal Nerve.
Vagus Nerve.
Accessory Nerve.
Hypoglossal Nerve.
Materials and Methods:



SD rats w/ weights of 180g-305g used.
Recombinant human IGF-1 used (96%
homology w/ rat IGF-1).
[125I]-IGF-1 (w/ less than 1% unbound
iodine) was ultrafiltrated x2 prior to each
experiment to remove unbound iodine.
Materials and Methods
(cont.):



Activity in cpm/µl was determined by a
gamma counter in tripicate.
Biotinylated IGF-1(2.3 mol biotin/mole
IGF-1) was used.
Catheters were implanted into the
abdominal aorta and/or the femoral vein
for blood sampling.
Materials and Methods
(cont.):


IN administration: Rats were anesthetized and
placed in a supine position. Total of 50 μl total
of [125I]-IGF-1 solution (5.0 nmol, 143 mcg
IGF/kg of body weight, specific activity of 3.5
Ci/mmol) was administered via pipette by drops
alternating between nostrils over 18.5 min.
IV administration: Rats placed in supine
position and 500 µl [125I]-IGF-1 (0.011 nmol
IGF-1, 0.33 mcg/Kg, 1900 Ci/mmol) injected via
femoral vein over 1-2 min.
Materials and Methods
(cont.):


Blood sampled Q5min from abdominal
aorta in both groups.
Animals transcardially perfused w/ saline
then fixed w/ mixture of glutaraldehyde
and paraformaldehyde approximately 30
min after start of administration.
Materials and Methods
(cont.):



One group of IN administered rats was allowed
to recover from anesthesia and blood was
sampled at 0.5, 2, 6, and 24 hrs after the start of
administration.
One group of IN administered rats (52 µl, 8.4
nmol, 290 mcg/Kg, specific activity= 1.6
Ci/mmol) were used to sample blood and CSF.
Post fixation, superficial and deep cervical
lymph nodes were dissected as well as dura
mater from the brain and blood vessels.
Materials and Methods
(cont.):



Fixed brain was serial sectioned (1mm) w/ a
coronal precision rat brain matrix.
Other areas of the brain were taken via corers (12 mm) using a micropunch technique.
Autoradiography was performed on two groups:
High specific activity IN (2100 Ci/mmol) and
Low specific activity IN (4.0 Ci/mmol).
Materials and Methods
(cont.):


Immunohistochemistry and Immunoblotting was
performed on two groups of IN administration:
60μl biotinylated IGF-I or control (PBS).
Animals were sacrificed and fixed after 24-28
min after the start of the administration.
Then brain tissue was sectioned and later
incubated w/ a 1:1000 dilution of a murine
monoclonal HRP-labeled antiphosphotyrosine
antibody to visualize tyrosine phosphorylated
proteins (by using metal enhanced DAB).
Materials and Methods
(cont.):

Immunobloating was performed using a
monoclonal antibody against
phosphotyrosine.
Conclusion:

Intranasal administration of therapeutic
proteins for the treatment of
neurodegenerative disorders may
overcome the limitations on the use of
neurotrophic factors by providing a safer,
more effective method of drug delivery to
the brain.