novel approach to deliver drugs across the blood-brain ... boer.pdf · proteins killing tumour...

Proteins killing tumour cells, Lorentz Workshop, Leiden - 2007

Novel approach to deliver drugs across the blood-brain barrier to the brain

A(Bert).G. de BoerBBB Research Group

Division of Pharmacology, LACDR University of Leiden, the Netherlands

Academic partner of to-BBB technologies BV


agenda

• introduction• the blood-brain barrier issue• strategies to bypass the BBB• receptor mediated transport• CRM197 technology

- (biopharmaceutical) drug delivery- pDNA delivery


Many patients with CNS diseases that are in need to be treated

• no centrally-active neuroprotective or neuroregenerative biopharmaceutical drugs on the market

- stroke, tumours, storage diseases, Alzheimer, Parkinson, Multiple Sclerosis, etc.

• largely under-developed and under-exploited area

• annual health care costs >> billions US$


I. neurodegeneration / neuroinflammation (Stroke, Alzheimers, Parkinsons, ALS, MS)- protein drugs

(growth factors, antibodies, enzymes) - gene / antisense therapy- small molecules

II. lysosomal storage diseases- enzyme replacement therapies- gene therapy

III. brain tumors / metastasis- anti-cancer drugs- protein drugs

(antibodies, cytokines)- gene / antisense therapy

applications of targeted brain delivery


direct administration of “biopharmaceutical” drugs is too local

for the treatment of human CNS disorders, breaching theBlood-Brain Barrier by direct injections / implants / waifers / gels / convection-enhanced delivery into the brain parenchyma or ventricles will only be partially effective:

– in AD and stroke large, global brain areas are affected – glioma’s are highly infiltrating, diffusely growing tumors– in PD and HD multiple brain areas are affected– in neuropathic pain, specific targets on sensory

neurons throughout the entire brain require modulation

..… therefore global brain drug delivery!

Alzheimer glioma

(Krewson, Brain Res. 1995)

Huntington(Yan, Exp Neurol. 1994)


agenda




strategies to cross the BBB

• BBB disruption (neurotoxic)• non-disruptive blood to brain delivery

– 20% of cardiac output goes to the brain– 600 km of capillaries– 20 m2 surface area– 1 km blood vessel per cm3

– “every neuron has its own capillary”

25 μm

0.6 μm

25 μm


agenda




crossing the Blood-Brain Barrier

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

blood

brain

continuousbasal membrane

+ ++

+

+

+

-- - - -- --

---

--- -

V.adsorptivemediated

endocytosis

BCEC

brain cells, like (the endfeet of) astrocytes, pericytes and neurons

VI.receptormediated

endocytosis

Y

Y

Y

Ytight junction

III.paracellular hydrophilic

diffusion

II.efflux

pumps (Pgp)

I.transcellular

lipophilicdiffusion

metabolism

IV.carrier

mediatedendocytosis

I--------------------------- small molecules ------------------------------------II--- biologicals ---I

strategies to bypass the BBB

(Abbott, Mol. Med. Today, 1996)


agenda




receptor-mediated endo(trans)cytosis:

proven CNS drug delivery mechanism of large payloads(small molecules, proteins, liposomes, DNA)

OX-26 examples (rodent transferrin receptor MAb)

(Pardridge, Nat.Rev.Drug,Discov. 2002)


(Pardridge, Adv.Exp.Med.Biol. 2002)


MAb’s to insulin or transferrin receptors- antibodies are “meant to stick” to its antigen

(unlike receptor-ligand interactions)

- interaction with either essential brain iron homeostasis or brain insulin signaling

ligands for LDL related protein receptor (LRP1/2)- LRP1/2 are multiligand scavenger and multiligand

signaling receptor at the BBB

- melanotransferrin (p97) activates plasminogen and co-localizes with beta-amyloid

competing receptors carry potential unwanted interactions,difficult to develop for human application:



agenda




blood

brain

carrierprotein

drug

transport receptor

safe & human applicablecarrier protein

2B-Trans™ technology


CRM197 transport receptor

diphtheria toxin receptor = membrane-bound (pro)HB-EGF

(Louie, Mol. Cell, 1997)


CRM197 carrier protein

CRM197; non-toxic mutant diphtheria toxin

• human applicable

– CRM197 - polysaccharide conjugate vaccines have already been marketed for many years

– CRM197 has been given systemically to advanced cancer patients to scavenge soluble growth factor

• proven excellent safety profile• proven mechanism of action• known manufacturing process

(Louie, Mol. Cell, 1997)


high constitutive expression in brain onblood vessels, neurons and gliahigh level of expression on muscle,heart, lymphocytes & spleen, kidneyand testes

0 1 2 3 4 5 6

Skeletal muscle

Brain

Heart

Liver

Spleen

Pancreas

Kidney

Testis

(arbitrary unit)Relative expression (mRNA)

Protein expression in brain (HB-EGF MAb)

but: strongly upregulated under inflammatory disease conditions disease induced targeting

CRM197 transport receptor

(Hayase et al., Brain Res. 1998)

(Abraham, Biochem.Biophys.Res.Comm. 1993)


I. specific binding toproHB-EGF / DTR

(KD 10-8-10-9 M)

II. receptor-mediated endocytosis of

CRM197-conjugate

III. pH shift-inducedreceptor dissociation

and change in proteinconformation

IV. endosomalescape

V. transcytosisacross the BBB

by diffusion

CRM197 conjugate or rec. fusion protein

BBB endothelial cell

CRM197 coated,drug-loaded liposome

BLOOD BRAIN

CRM197 conjugatedpolyplex of plasmid

CRM197 technology modalities


CRM197 technology in drug delivery

high and specific cellular uptake of CRM197

&no uptake of HRP or free label

DAPI/FITC (merge)

HRP-FITC FITC only untreated


PEGylated CRM197-liposomes

LLC-PK1 cells

hamster brain

LLC-PK1 cells


agenda




CRM197 technology: pDNA delivery


• Polyethylene glycol (PEG) 5000 dalton: spacer, water soluble, low immunogenicity

• PEI branched 45000 dalton (strong positive charge)

• CRM197 65000 dalton

CRM197

PEG PEI

CRM-PEG-PEI system


Transfection of U87 cells with pGFPby CRM197 technology

Number of GFP positive U87 cells

0.00

5.00

10.00

15.00

20.00

25.00

30.00

CRM-PEG-PEI HRP-PEG-PEI CRM-PEG-PEI (free CRM)

Posi

tive

cells

(% to

tal c

ell

num

ber)


CRM197-polyplexes deliver the Apoptin-gene into tumour cells to induce apoptosis

Cytoplasma &

nucleus

Apoptosis

Apoptin-gene delivery into tumour cells


Efficacy of CRM197- targeted Apoptin-gene delivery into tumour cells

Day

5

Day

2

DAPI FITC-MAb-Ap Merge

Apoptin-gene delivery into tumour cells


Conclusions• non-viral cell transfection mechanism for “biopharmaceuticals” and small

molecular drugs (liposomes)• specific receptor-mediated cellular uptake (targeted delivery)• endogenous endosomal escape mechanism• systemic delivery• disease-specific targeting

– passive; by enhanced retention through leaky blood vessels– active; by targeting to over-expressed receptors

• human applicable carrier protein• applicable in Apoptin-gene delivery

Application of CRM197 technology


RNAi delivery projects & applications (liposomal and PEI) in:

- HIV/neuroaidsProf.dr. B. Berkhout

plasmid delivery projects & applications(liposomal and PEI) in:

- stroke

- glioblastomaProf.dr. M.H.M. Noteborn

- Alzheimer’s disease

- Enzyme replacement therapyProf. Bill Sly (St. Louis)Prof. Bill Banks (St. Louis)

CRM197 technology in DNA & RNAi delivery


BBB Research Group, Div. Pharmacology, LACDR, Univ. Leiden:

Jaap RipGeert Schenk

Francesca MancaOlivier de Graaf

Hendrik KommerieAriane de Groot

Kayi ChanJesse van der Hoven

Collaborator Div. Medical Pharmacology,LACDR, Univ. Leiden:

Erno Vreugdenhil

Biological ChemistryLIC, Univ. Leiden:

Mathieu NotebornPatrick Voskamp

to-BBB technologies BV:Pieter Gaillard

Chantal AppeldoornRick DorlandJoan Knegten

acknowledgements

novel approach to deliver drugs across the blood-brain ... boer.pdf · proteins killing tumour...

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