Download Understanding Oral Bioavailability Challenges Associated With

AAPS
Chicagoland Pharmaceutical Discussion Group
Understanding Oral Bioavailability Challenges
Associated With Poorly Soluble Compounds That
Are Also Effluxed or Metabolized
Ming Hu, Ph.D.
College of Pharmacy
University of Houston
Houston, TX 77030
[email protected]
832--842832
842-8320
OUTLINE
¾
Overview of Gut and Liver Biology and Physiology
¾
Barriers to Oral Bioavailability
¾
Challenges Associated with Poorly Soluble Compounds
¾
Concluding Remark
OUTLINE
¾
Overview of Gut and Liver Biology and Physiology
¾
Barriers to Oral Bioavailability
¾
Challenges Associated with Poorly Soluble Compounds
¾
Concluding Remarks
OVERVIEW
¾
Intestinal and Gut Biology and Physiology
¾
Overview of Intestinal and Hepatic Enzymes and
Transporters
¾
Models Useful for Study of Intestinal and Hepatic
Enzymes and Transporters
Physiology:
The Digestive System
Human Physiology, 1998
Intestinal Barrier:
I t ti l Epithelium
Intestinal
E ith li
(LUMEN)
Intestinal Epithelium
apical
basolateral
(LUMEN)
Intestinal Epithelium
apical
basolateral
(LUMEN)
The Tight Junction
(LUMEN)
Summary of GI Tract Features
From Physiological Pharmaceutics: Davis, 2002
Liver Biology and Physiology
From Drug Metabolism by L. Gan
Liver Lobule
9/20/2013
Bar = 250 nm
12
Hepatocytes
¾
Hepatocytes are exceptionally active in synthesis and
transport. An ultra structural examination of
hepatocytes reveals bountiful quantities of both rough
and smooth endoplasmic reticulum.
reticulum. In contrast to most
glandular epithelial
g
p
cells that contain a single
g Golgi
g
organelle, hepatocytes typically contain many stacks of
Golgi membranes. The ultra structural appearance of
hepatocytes reflects their function as metabolic
superstars..
superstars
- Robert Bowen
9/20/2013
13
Blood Vessels Vs
Vs. Cannicular
9/20/2013
14
Bile Cannicular
9/20/2013
15
Transport
p Pathways
y and Mechanisms
Luminal pH
pHd = 6.0
Transport
Paracellular
Intracellular ppH
pHi=7.4
Active
Diffusion
Active
F ili
Facilitated
d
Parent
Diffusion
Facilitated
Metabolite
Serosal pH
pHr = 7.4
Uptake and Efflux Transporters
FDA DDI Guidance
G id
2012 Draft
D f
Uptake Transporters
Ê
Ê
Ê
Ê
Ê
Ê
Amino acids
Small peptides
(oligopeptides)
Nucleotides
OATPs
OCT
Others
Peptide Transporter (PepT1) Expression
Large quantities in duodenum, jejunum, ileum,
nearly absent (<10% of small intestine) from
colon in humans. (1. Drug Metab Dispos. 2007
35(8):1333--40. 2. Drug Metab Dispos. 2007
35(8):1333
35(4):590--4 )
35(4):590
™ High expression level in duodenum, jejunum and
ileum (used whole ileum)
ileum), but low expression in
colon. (Peptides. 2006 27(4):85027(4):850-7)
™
Drugs Transported by PEPT1
Pflugers Arch - Eur J Physiol (2004) 447:610–618
Efflux Transporters
P-Glycoprotein, MDR1 (other MDRs in
other species)
p
)
Ê BCRP
Ê MRPs (1
(1, 22, 33, 44, 55, 66, 7)
Ê BSEP
Ê MATEs
Ê OATs
Ê
P-glycoprotein
Ê
Ê
Ê
Victor Ling, 1976
Design and development of
inhibitors to enhance cancer therapy
in vivo has been the dreams of
newly four generations of scientists.
If a drug can be made, Dr. Ling will
receive a Nobel price for medicine.
Ê
Ê
Ê
Ê
Ê
First crystal structure of binding
subunit, published in 1998
First bacteria crystal structure of a
model protein,
protein 2002
First mammalian crystal structure
published in 2009
Countless papers of high quality
Billions of dollars and tens of
thousands of hours of intellectual
pursuit.
S G Aller et al. Science 2009;323:1718-1722
Binding of novel cyclic peptide P-gp inhibitors.
Published by AAAS
Fig. 4. Model of substrate transport by P-gp.
S G Aller et al. Science 2009;323:1718-1722
Published by AAAS
Metabolic Enzymes
Phase I Enzymes
CYPs
FMO
AO
Hydrolases (esterases)
Phase II Enzymes (Transferases)
UGTs
SULTs
GTs
Characteristics of Intestinal
E
Enzymes
andd Transporters
T
Ê Region
Region--Dependent
Ê Concentration
C
Concentrationi -Dependent
D
d
Rat Intestinal Efflux
Transporter Expression
Drug Metab Dispos. 2008 36(7):124936(7):1249-54.
LC-MS
LC
MS Based Assay
Fig.10. Amount of Bcrp in various rat tissues
BCRP
protein
(
(pmol/mg
25
2.5
2
15
1.5
MRM 1
MRM 3
1
05
0.5
0
Liver WT Liver KO Duo WT Duo KO
Jej WT
Jej KO
iLe WT
iLe KO Lung WT Lung KO
Amount of M
A
Metabolite E
Excreted
(nm
mol/30min)
Region-Dependent:
Region
Dependent: MRP2
14
12
10
8
6
4
2
0
Intestinal Excretion of Genistein Conjugates
Wistar
Duodenum
Jejunum
Ileum
TR
TR-
Colon
Region-Dependent Expression
Panel C: Microsomal Rates
Rattes of
Glucuronidation
(nmol/m
min/mg of
pro
otein)
4.0
*
3.0
20
2.0
*
*
*
1.0
0.0
Duodenum
Jejunum
Ileum
Colon
Am
mount of Conjugates Excreted
(nmol/30min
n)
Panel B: Excretion
20
*
*
Duodenum
Jejunum
*
15
10
5
0
Ileum
Colon
Wang et al, DMD, 34(11):1837-48, 2006
Summary Features of Enzymes and
T
Transporters
i Intestine
in
I
i
¾
Transporter:
¾
¾
¾
Enzymes:
¾
¾
¾
Region-specific (or dependent) expression
Region((“absorption
absorption window”)
window )
Polarized (apical vs. basolateral)
Region
R
Regioni -Specific
S ifi Expression
E
i
Membrane--bound vs. Cytosolic
Membrane
Excretion:
c et o :
¾
¾
Region-Specific
RegionApical vs. Basolateral
Summary Features of Enzymes and
T
Transporters
i Liver
in
Li
¾
Transporter:
¾
¾
Enzymes:
¾
¾
Polarized (apical vs. basolateral)
Membrane--bound vs. Cytosolic
Membrane
Excretion:
¾
Apical vs. Basolateral
Models and Experimental Systems
Physicochemical: log P, log D, solubility,
pKa, PAMPA and etc.
Ê Biologically based models:
Ê
Fidelity
Cost
¾
¾
¾
¾
¾
¾
Human PK
Animal PK
In situ
Excised tissue
Cell culture
M b
Membrane
vesicles/Microsomes
i l /Mi
Resolution
Speed
Epithelial Cell Culture Model
AP Side
(Lumen)
BL Side
(Serosal)
B to A
A to B
Apical to Basolateral Transport: Absorptive Transport
Basolateral to Apical Transport: Efflux Transport
The Perfused Rat Intestinal Model
0.1-0.4 ml/min
Heated to 37oC
Perfusion
Pump
Perfusion
Solution
Perfusate
D J I C
Control
Four site simultaneous perfusion model of rat intestine
Four-site
Flow rate may
y be changed
g to increase absorption/metabolism
p
during single pass perfusion.
OUTLINE
¾
Overview of Gut and Liver Biology and Physiology
¾
Barriers to Oral Bioavailability
¾
Challenges Associated with Poorly Soluble Compounds
¾
Concluding Remarks
Barriers to Oral Bioavailability
Solubility and Dissolution
¾ Absorption
¾ Excretion
¾ Metabolism
¾ Transport
p
to Specific
p
Targets
g
¾
Bioavailability Barriers
Intestine
Liver
Target
1
Systemic
Portal vein
2
4
Blood
?
3
?
3
4
4
?
Bypass
Hepatocytes
4
Bile
5
Importance of Solubility
Ê Vabs
=PSC
Ê Mabs = P S C TR
C: Mass Driving Force
Ê
Ê
F passive
For
i diffusion,
diff i P is
i dependent
d
d on partition
ii
coefficients, diffusion coefficients, and unstirred water
layer
y thickness.
For carriercarrier-mediated transport, P is dependent on Jmax
and Km of the transporter, and concentration. P
decreases with C when C approaches Km.
Transport
p Pathways
y and Mechanisms
Luminal pH
pHd = 6.0
Transport
Paracellular
Intracellular ppH
pHi=7.4
Active
Diffusion
Active
F ili
Facilitated
d
Parent
Diffusion
Facilitated
Metabolite
Serosal pH
pHr = 7.4
Oral Absorption Pathways:
Nutrient Carriers
Variety: amino acids, glucose, peptides
peptides,,
nucleotides,, nucleobases,, vitamins,, etc.
¾ Variable capacity: Km ranging from
several μM to several mM
¾ Structure: typically hydrophilic molecules
with nutrient like structures
¾ Stringent: critical structure motif needed
¾
Other Drug Carriers
Organic Anion Transporters
Ê Organic Anion Transporting Peptides
Ê Organic Cation Transporters
Ê Monocarboxylic
M
b
li Acid
A id T
Transporters
Ê
Excretion
¾ Efflux
transporters were first recognized as a normal
intestinal functions about 2 decades ago.
ƒ
Many efflux transporters recognize small hydrophobic
molecules, especially those with planar structures.
¾ Efflux
transporters were found to be necessary for
cellular production of phase II conjugates was
recognized
i d about
b
1 decade
d d ago.
Intestinal Excretion
¾
¾
Counter the Absorption of Lipophilic Substances
Efflux of Xenobiotics and Their Metabolites
¾
¾
¾
¾
P-Glycoprotein (MDRs)
BCRP
MRP and
MRPs
d Oth
Other ABC T
Transporters
t
Other Classes of Transporters
Efflux of Parent
L i l
Luminal
Paracellular
Efflux
Intracellular pH
ppHi=7.4
Enzy
yme
Efflux
S
Serosal
l
Hepatic Excretion
¾
¾
Eliminate Lipophilic Substances from Blood
Efflux of Xenobiotics and Their Metabolites
¾
¾
¾
¾
P-Glycoprotein (MDRs)
BCRP
MRP and
MRPs
d Oth
Other ABC T
Transporters
t
Other Classes of Transporters
Metabolism
First Pass Metabolism
Ê Phase I
Ê Phase II
Ê Interplay
I
l
Ê
Interplay
¾
Consequences of Interplay:
¾
¾
¾
Efficient production of phase II conjugates
Multiple or repeated exposure to the same enzymes
Recycling of xenobiotics (e.g., phenolics)
Interplay:
Excretion of Phase II Metabolites
L i l
Luminal
Excretion
Efflux
Enzy
yme
Intracellular pH
ppHi=7.4
S
Serosal
l
Interplay:
E
Excretion
ti off Parent
P
t and
d Metabolite
M t b lit
L i l
Luminal
Metabolism and Efflux
Efflux
Enzy
yme
Intracellular pH
ppHi=7.4
S
Serosal
l
Enteric and Enterohepatic
p
Recycling
y
g
LUMEN
Glucoside
Microflora
Microflora
Aglycone
LPH
MRP
Conjugates
BCRP
SGLT1
MRP2
OAT
β-glucosidase
WALL
Glucoside
Aglycone
Glucuronic Acid
Sulfate
OAT
MRP
?
bile
MESSENTARY BLOOD
Portal Vein
LIVER
MRP2
Heart
Saturation of Protein Mediated
Di
Disposition
iti (Metabolism
(M t b li or Efflux)
Effl )
Ê Vmet
met/ex
/ex
= Vmax C/(
C/(K
Km+C
+C))
Ê Mmet
C/(K
Km+C
+C)
C ) TR
met/ex
/ex = Vmax C/(
Ê
Ê
Ê
Ê
C: Saturation means higher concentration will not increase
rate of metabolism but may decrease it (substrate inhibition).
Saturation of efflux will result in favorable mass action.
C
Compound
d with
ith fast
f t permeability
bilit andd poor protein
t i binding
bi di may escape
metabolism or fair much better than predicted using microsomes.
Inward concentration gradient and outward protein concentration gradient
across the intestinal epithelium
p
favor uptake/absorption.
p
p
Recycling of parent compound in the gut due to efflux may increase the extent
of metabolism,
OUTLINE
¾
Overview of Gut and Liver Biology and Physiology
¾
Barriers to Oral Bioavailability
¾
Challenges Associated with Poorly Soluble Compounds
¾
Concluding Remarks
Biopharmaceutical
Cl ifi i System
Classification
S
Class I
Class II
High
Dissolution/Solubility
High
g Permeabilityy
Class III
Low
Dissolution/Solubility
High
g Permeabilityy
Class IV
High
i h
Dissolution/Solubility
L Permeability
Low
P
bilit
Low
Dissolution/Solubility
L Permeability
Low
P
bilit
Class II Drugs
•
•
•
Low Solubility
High Permeability
Lipophilic Drug Molecules That
Binds Often Selected via HTS.
Binds,
HTS
Dose Number = Dose/(Cs V)
Identification of Problems
Poor aqueous solubility, less than 5 μM
Ê Lower F at a higher dose in preclinical
species
Ê
™ Complications:
™ Efflux
™ Metabolism
™ Protein
Bindingg
Class IV Drugs
Poor solubility: small versus large dose
Ê Poor permeability: efflux vs.
vs uptake
Ê Poor solubility exasperates the efflux
problem and/or first pass metabolism
effects.
Ê
Identification of Problems
Ê
Ê
Ê
™
Low aqueous solubility, less than 5 μM
Highly
g y variable and poor
p
F at all doses in preclinical
p
species
High efflux ratio in efflux transporter overexpressed
cell culture models.
Complications:
Efflux
™ Metabolism
™ Food and/or fasting effects
™
Complication: Solubility and Efflux
Lower solubility means that higher potential
for efflux.
Ê Efflux inhibitors, with rare exception, have
not been successful in the clinic
clinic.
Ê Enhancement of solubility will alleviate or
even eliminate the problem.
problem
Ê
F will increase with dose.
Complication: Solubility and Metabolism
Lower solubility means less potential for
saturating
g intestinal metabolism.
Ê Intestinal inhibitors are sometimes
successful in the clinic if the enzymes are
not present in the liver.
Ê Enhancement of solubility will alleviate or
even eliminate the problem.
Ê
F will increase with dose.
Complication: Solubility and Microbiome
Ê
Ê
Ê
Lower solubility means that intestinal microbiome
will be exposed to the unabsorbed compounds
Microbiome can mediate highly different
metabolic pathway that may not be well conserved
across species.
i
Enhancement of solubility will exasperate the
problem
bl if absorption
b
ti remain
i largely
l
l incomplete.
i
l t
Example: Ginsenosides
R2O
R2O
OH
OH
20
0
3
HO
6
R1O
OR1
PPD type
PPT type
OH
R1O
OH
R1O
R2
Type C
R2
Type D
62
Ginsenoside Bioavailability
Ê
L exposure (oral
Low
( l bioavailability)
bi
il bilit ) off ginsenosides
i
id
Ê Rg3 oral administration in human
Ê
Ê
0.8 mg/kg:
g g below LOQ
Q
3.2 mg/kg: Cmax is 15.6 ng/ml
In Rat
Oral bioavailability (%)
Dose (mg/kg)
Rh2
4.7
1
Rg3(r)
< LOQ
100
CK
3.52
30
Re
0.16-0.28
1
Rb1
4 35
4.35
10
Rd
2.36
10
63
Identification of Problems
No phase I metabolism
Ê No phase II metabolism
Ê No stability problem
Ê Poor
P
solubility,
l bili less
l than
h 5 uM
M
Ê
Perm
meability (cm
m/s)
3.0E-05
2.5E-05
A to B
2.0E-05
B to A
1.5E-05
1 0E-05
1.0E
05
*
5.0E-06
*
*
*
0.0E+00
Efflux
ratio
(Pb-a / Pa-b)
28.5
1.0
1.2
I
Intracellular
r amounts (nmol/mg)
Cell Culture Studies
0.7
0.6
05
0.5
0.4
0.3
0.2
0.1
0
Cell Culture Studies
Rh2
Rh2
6.0E-05
B to A
4.0E-05
3 0E 05
3.0E-05
2.0E-05
1.0E-05
**
0.0E+00
MDCKII
MDR1-MDCKII MDR1-MDCKII
+20µM CsA
Inttracellular Conccentrations
(nmol/mgg)
5.0E-05
Permeabillity (cm/s)
08
0.8
A to B
**
0.7
0.6
0.5
0.4
0.3
0.2
0.1
*
0
MDCKII
MDR1-MDCKII
MDR1-MDCKII
+20µM CsA
Animal Studies
Plasma conccentrations ((µM)
P
10
FVB alone
Mdr1a/b -//
1
0.1
0.01
0
4
8
12
Time (hr)
16
20
24
Approaches
Ê
For highly permeable drugs, increase
solubility
y via nanoparticles,
p
, solid
dispersion, emulsion, excipients
excipients,, and other
pp
Hepatic
p
clearance
formulation approaches.
may still be a problem.
problem.
Ê
Cyclosporin A, Griseoflavin
Approaches
Ê
For poorly permeable drugs that are poorly soluble,
identify the role of efflux transporters (if any), and then
define approaches to the problems.
problems
Ê
Ê
Ê
If efflux transporters are involved, solubility increase will greatly
help. However, this may not solve rapid hepatic clearance
problem.
bl
If extensive metabolism is known, intestinal metabolism may be
inhibited but hepatic metabolism will remain a big problem.
If no efflux transporters are involved, then prospects are very poor.
Prodrugs are possible but often not viable based on business
considerations. This may work for second generation of drugs.
Phenolic Drugs
Many phenolic drugs are rapidly metabolized
Ê For highly potent drugs (effective in vivo
concentration in nM/pM range), phenolics
may not be as bad as feared
feared.
Ê Futile recycling may result in low
bioavailability but long halfhalf-life (many sex
hormones and SERMs such as raloxifene).
Ê Phase
Ph
2 metabolism
b li may be
b less
l variable.
i bl
Ê
Collaboration with Industry
Ê
Ê
Ê
Ê
Ê
Regional Absorption: Should we develop a
sustained release formulation ?
Solubility Enhancement: Would it really matter?
Model System Characterization: Is the model
reliable and reproducible?
Protein Drug Adsorption: How to minimize the
impact in clinical setting?
Protein quantitation: Is it ready for prime time in
ADME and PK?
OUTLINE
¾
Overview of Gut and Liver Biology and Physiology
¾
Barriers to Oral Bioavailability
¾
Challenges Associated with Poorly Soluble Compounds
¾
Concluding Remarks
Concluding Remarks
Ê
Ê
Compounds with poor water solubility and
high
g permeability,
p
y, as well as slow
metabolism or efflux is a prime candidate
for elaborate formulation efforts. The
caveat is that human disposition should be
well established to decrease the chance of
erroneous designation of low “metabolism”
or “efflux.”
Concluding Remarks
Ê
Compounds with poor solubility and
extensive efflux by
y intestinal but not hepatic
p
apical efflux transporters is a also a good
candidate for elaborate formulation efforts.
Ê
Question? Should we even consider the importance of
efflux transporters that are only expressed in the kidney?
Concluding Remarks
Ê
Compounds with poor solubility and
extensive efflux by
y both intestinal and
hepatic apical efflux transporters is a
g g candidate to develop,
p, and mayy
challenging
not deserve elaborate formulation efforts.
Ê
Question? Should we really develop poorly soluble
compounds that are pp-glycoprotein or efflux transporter
inhibitors (e.g., cyclosporin A like)?
Concluding Remarks
Ê
Compounds with poor solubility and
extensive metabolism by
y both intestinal and
hepatic apical enzymes is a challenging
p, and should be
candidate to develop,
abandoned unless it is highly potent and
g recycling.
y
g
undergo
Ê
Question? Could phenolic compounds that are highly potent
but extensively metabolized and recycled always succeed?
Concluding Remarks
Ê
Compounds with poor solubility and
extensive metabolism by
y only
y intestinal but
not by hepatic apical enzymes is a good
candidate to for elaborate formulation efforts
to increase its solubility.
Ê
Question? Do you know many phase II enzymes display
substrate inhibition kinetics, meaning lower rates at higher
concentration?
Concluding Remarks
Ê
Because intestinal microbiome will be
exposed
p
to unabsorbed compounds
p
and
intestinal microbiome structures appear to
correlate with human health,, FDA will
begin to mandate studies of a drug’s effect
on intestinal microbiome within a decade.
The tools should be ready then.
Acknowledgement
¾
Postdoctoral Researcher
¾
Ph.D. Students
¾
¾
¾
¾
¾
¾
¾
¾
¾
¾
¾
Grant
Dr. Jun Chen
Dr.. Song Gao
Dr
D B
Dr.
Beibei
ib i Xu
X
¾
¾
¾
Dr. Stephen Wang
D Zh
Dr.
Zhen Yang
Y
Dr. Zhu Wei
Dr. Kaustubh H Kulkarni,
Dr.
Dr Rashim Singh
Dr. Wen Jiang
Dr. Baojian Wu
¾
¾
¾
¾
¾
Ms. Terry Yin
Collaborator
¾
¾
¾
Senior Research Associate:
¾
NIH AT 000182
NIH AT 003203
NIH AT 005522
NIH GM 070737
NIH CA 087779
Dr. Ming You, Medical
College of Wisconsin
Dr.
Dr Joseph Petrosino,
Petrosino Baylor
College of Medicine
Dr. Zhongqiu Liu,
Guangzhou University of
Chi
Chinese
Medicine
M di i
Role of MRP2
Amountss Extcretted
(nmoll/30 min)
3.0
2.5
Wistar
Billary Genistein Conjugates
TR-
2.0
1.5
1.0
0.5
0.0
60
90
120
Time (min)
(
)
150