Nov 5, 2014
Symposium in AAPS2014 (San Diego)
What is new in oral delivery of biologics?
Mechanistic aspects, delivery platforms, and
clinical development with case studies.
Paracellular or transcellular?
Promising approaches for oral delivery
of macromolecules
Masuo Kondoh, Kiyohito Yagi
Graduate School of Pharmaceutical Sciences
Osaka University
Spiral progression
The philosopher Hegel（1770-1831）
Future
Present
Past
Nothing is lost or destroyed but elevated and
preserved as in a spiral.
The past will re-emerge with progress.
Motivation behind today’s talk
The philosopher Hegel（1770-1831）
Future
Present
Past
Nothing is lost or destroyed but raised up and
preserved as in a spiral.
The past will re-emerge with progress
Spiral progression of oral delivery of macromolecules
with progress in understanding the epithelial barrier
Outline of today’s talk
Future
1）The beginning of the spiral progression
・What the epithelial barrier was thought to be
・1st generation permeation enhancers
・Transporter- and receptor-mediated strategies
Present
Past
2) Recent spiral progression
・Current understanding of the epithelial barrier
・2nd generation permeation enhancers
・Tricellular tight junctions
Biologics
http://www.xconomy.com/seattle/2009/07/09/how-healthcare-legislationcan-ensure-patient-safety-and-spur-innovation/
Interface for oral delivery of biologics
Mucosal surface area per person
http://upload.wikimedia.org/wikipedia/commons/8/8f/Illu_epithelium.jpg
The beginning of the spiral progression
in enhancing absorption
Windsor and Cronheim, Nature, 4772, 263, 1961
EDTA enhanced mucosal absorption of heparin
However, the mode of action was unclear.
Epithelial barrier
TJ : tight junction
Before 1963
Tsukita and Furuse, Trend Cell Biol, 9, 268, 1999
After 1963
TJ
Absorption enhancers (of many)
Enhancer
EDTA
Sodium caprate
Sodium laurate
Year
1960s
1980s
1980s
PODTM technology (Oramed Pharm., Inc.)
Insulin
U.S. Phase 2a
Increased paracellular permeability
of possible toxic molecules
EDTA
Will launch in 2015
TPETM technology (Chiasma, Inc.)
Octreotide
Phase 3
Sodium caprylate
Eligen® technology (Emisphere Technol., Inc.)
Calcitonin, heparin, insulin
Phase 2
SNAC
ChronotropicTM technology (Dexcel Pharma Technol., Ltd.)
Insulin
Sodium glycocholate
GIPETTM technology (Merrion Pharma Technol., Ltd.) Increased paracellular permeability
Acyline, Heparin, Insulin, GLP-1
Sodium caprate
AxcessTM delivery system (Proxima Concept, Ltd.)
Insulin
Phase 2a
Permeation enhancers
of possible toxic molecules
Increased paracellular permeability
of possible toxic molecules
PeptelligenceTM technology (Enteris BioPharma, Inc.) Increased paracellular permeability
Calcitonin
Phase 3
Permeation enhancers
Ref) Choonara et al., Biotechnol. Adv., in press
of possible toxic molecules
TPE
technology (Chiasma, Inc.)
Octreotide
Absorption enhancers
Phase 3
Sodium caprylate
Eligen® technology (Emisphere Technol., Inc.)
Calcitonin, heparin, insulin
Phase 2
SNAC
ChronotropicTM technology (Dexcel Pharma Technol., Ltd.)
Insulin
Sodium glycocholate
GIPETTM technology (Merrion Pharma Technol., Ltd.) Increased paracellular permeability
Acyline, Heparin, Insulin, GLP-1
Sodium caprate
AxcessTM delivery system (Proxima Concept, Ltd.)
Insulin
Phase 2a
Permeation enhancers
of possible toxic molecules
Increased paracellular permeability
of possible toxic molecules
PeptelligenceTM technology (Enteris BioPharma, Inc.) Increased paracellular permeability
Calcitonin
Phase 3
Permeation enhancers
Ref) Choonara et al., Biotechnol. Adv., in press
Absorption
enhancer
TJ
of possible toxic molecules
Spiral progression continues
Enhancer
EDTA
Sodium caprate
Sodium laurate
Year
1960s
1980s
1980s
Problems with absorption-enhancers
１）Influx of non-drug solutes by opening TJ
２）Non-specificity
３）Cytotoxicity
Paracellular delivery
Transcellular delivery
TJ
Active targeted drug delivery systems
RCF, reduced folate transporter; PCFT, proton-coupled folate transporter
SMVT, sodium-dependent multivitamin transporter
Ref) Li et al., Expert Opin. Drug Deliv., in press
Active targeted drug delivery systems
Problems with ATDDS
１）Conjugation: loss of activity and affinity
２）Fusion: low bioavailability due to larger size
３）Carrier: size limitations
４）All: GI degradation of ligands
Currently no clinical studies
Ref) Li et al., Expert Opin. Drug Deliv., in press
How to resolve these problems?
Problems with absorption-enhancers
１）Influx of non-drug solutes by opening TJ
Paracellular delivery
２）Non-specificity
３）Cytotoxicity
Future
Problems with ATDDS
Present
１）Conjugation: loss of activity and affinity
２）Fusion: low bioavailability due to larger size
Past
Transcellular delivery
３）Carrier: size limitation
４）All: GI degradation of ligands
TJ
Spiral progression
The philosopher Hegel（1770-1831）
Future
Present
Progress in Barriology
From para to trans DDS
Past
1963：Identification of TJ
Spiral progression of oral delivery for macromolecules
with progress in understanding of the epithelial barrier.
Biological roles of TJs
1) Barrier separating the apical side and
the basal side
2) Compartmentalization of tissues
3) Maintenance of homeostasis in each tissue
by regulation of solute movement
Goal
keeper
Gate
keeper
Modulation of the TJ barrier
Will lead to the development of
novel tissue- and drug-specific delivery systems
Progress in TJ cell biology
TJ
Year
Event
1963
Identification of TJ
1964
Various CLDNs
Opening TJ-seal by EDTA
Occludin
1973
Identification of TJ strands
1982
Lipid micelle theory
1993
Identification of occludin
1998
Identification of claudin (CLDN)
1999 onwards
TJ-barrier function of CLDNs
2005
Identification of tricellulin
CLDNs, key components in TJ-seal formation
CLDN family
Epithelium
TJs
CLDN
・MW ~23 kDa
・A tetra-transmembrane protein
・Component of the TJ-seal
・27 members
・Tissue-specific expression
・Tissue-specific barrier function
CLDN-1, epidermis; CLDN-4, mucosal epithelium; CLDN-5, blood-brain-barrier
CLDN strands
Side-by-side
CLDNs-based paracellular routes
Size-selective
Charge-selective
Head-to-head
Trends Cell Biol, 2006
The CLDN technology will make a breakthrough
in paracellular drug delivery
from disrupting or opening TJs-seal to modulating TJs-seal
Classification of absorption-enhancers
1st generation absorption-enhancers (of many)
Absorption-enhancer
Possible mode of action
EDTA
Sequestration of Ca2+
Surfactants
Perturbation of the plasma membrane
Sodium caprate
Phospholipase C
2nd generation absorption-enhancers (of many)
Absorption-enhancer
Possible mode of action
Occludin EL peptide
CLDN EL peptide
C-CPE
Prevention of head-to-head interaction
Prevention of head-to-head interaction
CLDN-3/-4
Drug Discov Today, 2008
Dynamism of CLDN strands
Annealing
CLDN strands
min:sec
Bar= 1 mm
Breaking
CLDN strands
PNAS, 100, 3971, 2003
CLDN strands exhibit dynamic behavior
Curr Opin Cell Biol, 16, 140, 2004
Breaking/resealing CLDN strands may result in solute movement
Schematic illustration of CLDN-strands
1st generation enhancers
Disruption or opening TJs-seal
2nd generation enhancers
No disruption or opening TJs-seal
Safety evaluation of the CLDN strategy
CLDN-5 knockout analysis
Tracer assay
CLDN-5 +/+
Morphology of tight junction
CLDN-5 -/-
CLDN-5 +/+
Red, biotinylation reagent (443 Da)
CLDN-5 -/-
J Cell Biol, 161, 653, 2003
Influx of solutes (~800 Da) into brain
TJ with normal appearance
CLDN-5 knockdown analysis
CLDN-5
siRNA
24ｈ
48ｈ
72ｈ
Blue, Hoechst 33342 (562 Da)
Green, FITC-dextran (4400 Da)
J Gene Med, 10, 930, 2008
Control
siRNA
No apparent adverse effects during the 48 h period
Targets for 2 nd generation enhancers
Bicellular tight junction (bTJ)
Occludin
Claudin
Tricellulin
Angulin
（LSR）
Tricellular tight junction (tTJ)
CLDN-targeted mucosal absorption
C-CPE
・A receptor binding domain of Clostridium perfringens
enterotoxin
・The first identified CLDN binder and modulator
・Binding to CLDN-3 and -4 with high affinity
・The best characterized CLDN binder
J Biol Chem, 283, 268, 2008
20
15
10
5
0
0
0.05 0.10
C-CPE
Amount of absorbed FDs
(h·µg/ml)
Amount of absorbed FD-4
(h·µg/ml)
Jejunal absorption of FD
40 (mg/ml)
Sodium caprate
16
14
12
10
8
6
4
2
0
Vehicle
C-CPE 0.1 mg/ml
FD-4
FD-10
FD-20
FD-40
Mol Pharmacol, 67, 749, 2005
CLDN-3/-4 binder is a 400-fold more potent absorption enhancer
Mucosal absorption of a biologic
Plasma hPTH (ng/ml)
hPTH：human parathyroid hormone. MW 4000.
A clinically used peptide drug.
Injection route of admin. Biochem Pharmacol, 79, 1437, 2010
Nasal
3
Jejunal
3
Vehicle
300
Vehicle
CLDN binder
CLDN binder
0.2 mg/ml
0.2 mg/ml
4.0 mg/ml
2
200
1
1
100
30 60 90
Time (min)
0
120 0
30 60 90
Time (min)
120
Vehicle
CLDN binder
0.2 mg/ml
0.8 mg/ml
2
0
0
Pulmonary
0
0
30 60 90
Time (min)
120
CLDN binder enhanced mucosal absorption of a peptide drug
Effects of CLDN-specificity on jejunal absorption
C-CPE
・CLDN-3/-4 binder
m19
・A C-CPE mutant
・CLDN-1~-5 binder
Blue: positive charge
White: neutral
Red: negative charge
8
AUC values
Vehicle
C-CPE
m19
6
4
2
0
0
1
2
3
4
Time (h)
5
6
Absorbed FD-4 (µg・h/ml)
Plasma FD-4 (µg/ml)
Time-course
* p<0.05
20
15
10
5
0
Vehicle C-CPE
m19
Biomaterials, 33, 3464, 2012
CLDN specificity may control absorption-enhancing activity
The first angulin binder, angubindin-1
Tricellular TJ
The first
tTJ component
A determinant factor
for the localization of
tricellulin
Tissue Barriers, 2, e28755, 2014
Angulin binder
Cell numbers
Angubindin-1
28 kDa
EpH4 cells
(Angulin-1)
LSR-kd cells
Angulin-2/
LSR-kd cells
Angulin-3/
LSR-kd cells
Our unpublished data
Fluorescence intensity
Angubindin-1 is an angulin-1 and -3 binder
Immuno-fluorescent analysis of angubindin-1
Angulin
Angubindin-1
Merge + DAPI
EpH4 cells
(Angulin-1)
Angulin-2/
LSR-kd cells
Angulin-3/
LSR-kd cells
Our unpublished data
Angubindin-1 is a tricellular TJ binder.
Effects of angubindin-1 on localization
of biotin reagent passage
Vehicle-treated cells
X-Z image
X-Y image
Y-Z
image
Angubindin-1-treated cells
Merge:
X-Z image
Y-Z
image
X-Y image
mZO-1
Biotin DAPI
mZO-1
Biotin
DAPI
Our unpublished data
Angubindin-1 is a modulator of tricellular TJs
Cut off sizes in TJs
TJs
Tricellular TJ
Tissue Barrier, 2, e28755, 2014
Cut off sizes in TJs
0.1
1.0
Diameter (nm)
10
Compr Physiol, 2, 1819, 2012
Take home messages
Transcellular technology is in the translational stage.
1) Clinical evaluation ~from bench to bed side~
2) Overcoming future problems encountered in the beginning
of clinical studies
3) Issues with conjugation of ligand and biologics
Paracellular technology using 1st generation enhancers
is in the clinical stage.
Paracellular technology using 2 nd generation enhancers
is at the stage of basic research.
1) Safety evaluation
2) Development of bi- or tri-specific claudin binders
3) Combination of bTJ binder and tTJ binders
Spiral progression
of our TJ projects
Spiral progression
The philosopher Hegel（1770-1831）
2026
Future
Present
2014
2002
Past
Acknowledgements
Freie Universitat and Humboldt
Prof. Michael Fromm
Dr. Susanne M. Krug
National Institute of Health Sciences
Dr. Akiko Watabe-Ishii
Dr. Minor Tada
Showa Pharmaceutical University National Institute of Infection Diseases
Dr. Masayoshi Fukasawa
Prof. Yoshiaki Watanabe
Dr. Makiko Fujii
Ehime University
Dr. Naoya Koizumi
Prof. Tatsuya Sawazaki
Dr. Hiroyuki Takeda
Asubio Pharma. Co. Ltd.
Dr. Hiroshi Uchida
Dr. Takeshi Hanada
The University of Tokyo
Prof. Takao Hamakubo
Osaka University
Prof. Yasuhiko Horiguchi
Prof. Tadayuki Uno
Dr. Hiroshi Aoyama
Kobe University
Prof. Mikio Furuse
Funding
Acknowledgements
Thank you for your attention!!
当グループのパイプライン
Binder創製
低分子化合物
論文
POC
特許申請
CL-3, CL-4
粘膜吸収促進
国内・PCT
論文
CL-3, CL-4
粘膜ワクチン 国内・PCT
論文
CL-3, CL-4
癌治療
国内・PCT
論文
CL-1~-5
粘膜吸収促進 国内・PCT
論文
Angulin-1/-3
粘膜吸収促進 国内・PCT
論文
CL-1
HCV感染阻害 国内・PCT
論文
CL-4
癌診断・治療
論文
CL-3, CL-4
CL-1, CL-2
CL-4, CL-5
ペプチド
抗体
CL-2
CL-5
Occludin
国内・PCT
補足資料2
低分子claudin binder創製系
Claudin binderのハイスループットスクリーニング（HTS）系
TR-FRET
XL665-anti GST
アルファスクリーン
Acceptor
655 nm
ドナービーズ
アクセプター
1O
2
ビーズ
励起光
(680 nm)
Claudin
Eu(K)-anti His
Donor
CL
1O
2
発光シグナル
（520-620 nm）
Claudin
binder
620 nm
337 nm
1O
2
発光シグナル
（520-620 nm）
使用予定のライブラリ
化合物ライブラリ
文科省創薬等PF事業
理研化合物ライブラリ
特殊環状ペプチドライブラリ
文科省創薬等PF事業
ファージライブラリ
scFv, ペプチド